Methods for transduction and cell processing

ABSTRACT

Provided are methods, systems, and kits for cell processing, e.g., for therapeutic use, such as for adoptive cell therapy. The provided methods include transduction methods, in which cells and virus are incubated under conditions that result in transduction of the cells with a viral vector. The incubation in some embodiments is carried out in an internal cavity of a generally rigid centrifugal chamber, such as a cylindrical chamber made of hard plastic, the cavity of which may have a variable volume. The methods include other processing steps, including those carried out in such a chamber, including washing, selection, isolation, culture, and formulation. In particular, the disclosure relates to method providing advantages over available processing methods, such as available methods for large-scale processing. Such advantages include, for example, reduced cost, streamlining, increased efficacy, increased safety, and increased reproducibility among different subjects and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.62/075,801 filed Nov. 5, 2014, entitled “Methods for Transduction andCell Processing,” and U.S. provisional application No. 62/129,023 filedMar. 5, 2015, entitled “Methods for Transduction and Cell Processing,”the contents of which are incorporated by reference in their entirety.

FIELD

The present disclosure relates to cell processing for therapeutic use,such as for adoptive cell therapy. The provided methods generallyinclude transduction methods, in which cells and viral vector particlesare incubated under conditions that result in transduction of the cellswith a viral vector. The incubation may be carried out in an internalcavity of a generally rigid centrifugal chamber, such as a cylindricalchamber made of hard plastic. The methods include other processingsteps, including those carried out in such a chamber, including washing,selection, isolation, culture, and formulation. In particular, thedisclosure relates to method providing advantages over availableprocessing methods, such as available methods for large-scaleprocessing. Such advantages include, for example, reduced cost,streamlining, increased efficacy, increased safety, and increasedreproducibility among different subjects and conditions.

BACKGROUND

Certain methods are available for cell processing, including large-scalemethods and methods for use in preparation of cells for adoptive celltherapy. For example, methods for viral vector transfer, e.g.,transduction, selection, isolation, stimulation, culture, washing, andformulation, are available. Available methods have not been entirelysatisfactory. Improved methods are needed, for example, for large-scaleprocessing, e.g., transduction, of cells for adoptive cell therapy. Forexample, methods are needed to improve efficiency and reproducibility,and to reduce time, cost, handling, complexity, and/or other parametersassociated with such production. Among the provided embodiments aremethods, systems, and kits addressing such needs.

SUMMARY

Provided are methods for cell processing, such as for transfer of viralvectors and/or immunoaffinity-based selection of cells. In someembodiments, the cells are for use in cell therapy, such primary cellsprepared for autologous or allogeneic transfer, e.g., in adoptive celltherapy. The methods may include additional cell processing steps, suchas cell washing, isolation, separation.

In some embodiments, the methods are carried out by incubating, in avessel, such as an internal cavity of a centrifugal chamber, acomposition (deemed an input composition), which contains cells andviral vector particles, the viral particles containing a recombinantviral vector, thereby generating an output composition that contains aplurality of the cells transduced with the viral vector. The centrifugalchamber typically is rotatable around an axis of rotation. The axis ofrotation in some embodiments is vertical. The chamber typically includesan end wall, a side wall extending from the end wall, such as asubstantially rigid side wall, and at least one opening, such as aninlet/outlet or an inlet and an outlet. At least a portion of the sidewall generally surrounds the internal cavity. The at least one opening(e.g., the inlet/outlet or the inlet and the outlet) is capable ofpermitting intake of liquid into the internal cavity and expression ofliquid from the cavity. The at least one opening in some embodiments iscoaxial with the chamber and in some embodiments is in an end wall ofthe chamber. The side wall may be a curvilinear, e.g., cylindrical orgenerally cylindrical.

In some embodiments, the methods include incubating, in an internalcavity of a centrifugal chamber, an input composition containing cellsand viral particles containing a recombinant viral vector, wherein saidcentrifugal chamber is rotatable around an axis of rotation and includesan end wall, a substantially rigid side wall extending from said endwall, and at least one opening, at least a portion of said side wallsurrounding said internal cavity and said at least one opening beingcapable of permitting intake of liquid into said internal cavity andexpression of liquid from said cavity, wherein the centrifugal chamberis rotating around said axis of rotation during at least a portion ofthe incubation and the method generates an output composition containinga plurality of the cells transduced with the viral vector.

In some embodiments, the centrifugal chamber further includes a movablemember, such as a piston. In such embodiments, the internal cavity isgenerally one of variable volume, e.g., a cavity of variable volumedefined by the end wall, the side wall, and the movable member, e.g.,the piston, such that the movable member is capable of moving within thechamber (such as axially within the chamber) to vary the internal volumeof the cavity. In some embodiments, liquid is moved in and out of thechamber alternatively by way of a pump, syringe, and/or motor, or otherdevice for intake and expressing liquid or gas, which for example pullsliquid from the cavity and/or pushes liquid in, while the volume of thecavity itself remains constant.

In some embodiments, the methods include incubating, in an internalcavity of a centrifugal chamber, an input composition containing cellsand a viral particle containing a recombinant viral vector, saidcentrifugal chamber being rotatable around an axis of rotation andcomprising an end wall, a substantially rigid side wall extending fromsaid end wall, and at least one opening, wherein at least a portion ofsaid side wall surrounds said internal cavity and said at least oneopening is capable of permitting intake of liquid into said internalcavity and expression of liquid from said cavity, wherein thecentrifugal chamber is rotating around the axis of rotation during atleast a portion of the incubation, the total liquid volume of said inputcomposition present in said cavity during rotation of said centrifugalchamber is no more than about 5 mL per square inch of the internalsurface area of the cavity and the method generates an outputcomposition comprising a plurality of the cells transduced with theviral vector.

The chamber may comprise two end walls. In some such embodiments, oneend wall together with other features defines the internal cavity, whilethe other is outside of the cavity. In some embodiments, the cavity isbound by both end walls.

The at least one opening may comprise: an inlet and an outlet,respectively capable of permitting said intake and expression, or asingle inlet/outlet, capable of permitting said intake and saidexpression.

Typically, the incubation is carried out at least in part under rotationof the chamber, such as under centrifugal force or acceleration. Thus,the methods in some embodiments further include effecting rotation ofthe centrifugal chamber, such as around its axis of rotation, during atleast a portion of the incubation.

In some of any such embodiments, said rotating includes rotation at arelative centrifugal force (RCF) at an internal surface of the side wallof the cavity and/or at a surface layer of the cells of greater than ator about 200 g, greater than at or about 300 g, or greater than at orabout 500 g. In some of any such embodiments, said rotating includesrotation at a relative centrifugal force at an internal surface of theside wall of the cavity and/or at a surface layer of the cells that is:at or about 1000 g, 1500 g, 2000 g, 2100 g, 2200 g, 2500 g or 3000 g; orat least at or about 1000 g, 1500 g, 2000 g, 2100 g, 2200 g, 2500 g, or3000 g. In some of any such embodiments, said rotating includes rotationat a relative centrifugal force at an internal surface of the side wallof the cavity and/or at a surface layer of the cells that is: between orbetween about 1000 and 3600, 1000 and 3200, 1000 and 2800, 1000 and2000, 1000 and 1600, 1600 and 3600, 1600 and 3200, 1600 and 2800, 1600and 2000, 2000 and 3600, 2000 and 3200, 2000 and 2800, 2800 and 3600,2800 and 3200, 3200 and 3600, each inclusive; or at least at or about2000 g, 2100, 2200 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g or 3600 g;or at or about 2000 g, 2100 g, 2200 g, 2400 g, 2600 g, 2800 g, 3000 g,3200 g or 3600 g.

In some of any such embodiments, the at least a portion of theincubation during which the chamber is rotating is for a time that is:greater than or about 5 minutes, such as greater than or about 10minutes, greater than or about 15 minutes, greater than or about 20minutes, greater than or about 30 minutes, greater than or about 45minutes, greater than or about 60 minutes, greater than or about 90minutes or greater than or about 120 minutes; or between or betweenabout 5 minutes and 60 minutes, 10 minutes and 60 minutes, 15 minutesand 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes or45 minutes and 60 minutes, each inclusive.

In some embodiments, the input composition (or the number of cells) inthe cavity during the incubation, e.g., at any one time or during theentire incubation, and/or processed by the methods, includes at or aboutor at least about 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸ or 5×10⁸ of thecells.

In some of any such embodiments, said input composition in the cavitycontains at least at or about 1×10⁷ of said cells, at least at or about2×10⁷ of said cells, 3×10⁷ of said cells, at least at or about 4×10⁷ ofsaid cells, at least at or about 5×10⁷ of said cells, at least at orabout 6×10⁷ of said cells, at least at or about 7×10⁷ of said cells, atleast at or about 8×10⁷ of said cells, at least at or about 9×10⁷ ofsaid cells, at least at or about 1×10⁸ of said cells, at least at orabout 2×10⁸ of said cells, at least at or about 3×10⁸ of said cells orat least at or about 4×10⁸ of said cells.

In some embodiments, the internal surface area of the cavity is at leastat or about 1×10⁹ μm² or 1×10¹⁰ μm², and/or the length of the side wallin the direction extending from the end wall is at least about 5 cmand/or at least about 8 cm; and/or the internal cavity has a radius ofat least about 2 cm at least one cross-section.

In some embodiments, the input composition includes at least or about 1infectious unit (IU) per one of the cells, at least or about 2 IU perone of the cells, at least or about 3 IU per one of the cells, at leastor about 4 IU per one of the cells, at least or about 5 IU per one ofthe cells, at least or about 10 IU per one of the cells, at least orabout 20 IU per one of the cells, at least or about 30 IU per one of thecells, at least or about 40 IU per one of the cells, at least or about50 IU per one of the cells, or at least or about 60 IU per one of thecells. In some embodiments, the input composition includes at or about 1infectious unit (IU) per one of the cells, at or about 2 IU per one ofthe cells, at or about 3 IU per one of the cells, at or about 4 IU perone of the cells, at or about 5 IU per one of the cells, at or about 10IU per one of the cells, at or about 20 IU per one of the cells, at orabout 30 IU per one of the cells, at or about 40 IU per one of thecells, at or about 50 IU per one of the cells, or at or about 60 IU perone of the cells.

In some embodiments, the average liquid volume or maximum liquid volumeof the input composition, composition with viral vector particles andcells, and/or any liquid composition present in the cavity during theincubation is no more than about 10, 5, or 2.5 milliliters (mL) persquare inch of the internal surface area of the cavity during theincubation. In some embodiments, the maximum total volume of such liquidcomposition present in the cavity at any one time during the incubationis no more than 2 times, no more than 10 times, no more than 100 times,no more than 500 times or no more than 1000 times the total volume ofthe cells. In some embodiments, the total volume of cells is the totalvolume of a pellet of the cells. In some embodiments, the total volumeof cells is the volume of a monolayer of the cells, such as a monolayerof cells present on the internal surface in the cavity during rotationof the centrifugal chamber.

In some embodiments, the liquid volume of the input composition occupiesall or substantially all of the volume of the internal cavity during atleast a portion of the incubation. In other embodiments, during at leasta portion of the incubation, the liquid volume of the input compositionoccupies only a portion of the volume of the internal cavity, the volumeof the cavity during this at least a portion further comprising a gas,which is taken into the cavity, e.g., via said at least one opening oranother opening, prior to or during the incubation.

In some of any such embodiments, the liquid volume of said inputcomposition present in said cavity during said rotation is between orbetween about 0.5 mL per square inch of the internal surface area of thecavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in.

In some of any such embodiments, the maximum total liquid volume of saidinput composition present in said cavity at any one time during saidincubation is no more than 2 times, no more than 10 times, or no morethan 100 times, the total volume of said cells in said cavity or theaverage volume of the input composition over the course of theincubation is no more than 2, 10, or 100 times the total volume of cellsin the cavity.

In some of any such embodiments, the maximum volume of said inputcomposition present in said cavity at any one time during saidincubation or the average volume over the course of the incubation is nomore than at or about 2 times, 10 times, 25 times, 50 times, 100 times,500 times, or 1000 times the volume of a monolayer of said cells formedon the inner surface of said cavity during rotation of said chamber at aforce of at or about 2000 g at an internal surface of the side wall.

In some of any such embodiments, the liquid volume of the inputcomposition is no more than 20 mL, no more than 40 mL, no more than 50mL, no more than 70 mL, no more than 100 mL, no more than 120 mL, nomore than 150 mL or no more than 200 mL.

In some of any such embodiments, during at least a portion of theincubation in the chamber or during the rotation of the chamber, theliquid volume of the input composition occupies only a portion of thevolume of the internal cavity of the chamber, the volume of the cavityduring said at least a portion or during said rotation furthercomprising a gas, said gas taken into said cavity via said at least oneopening, prior to or during said incubation.

In some embodiments, the centrifugal chamber includes a movable member,whereby intake of gas into the centrifugal chamber effects movement ofthe movable member to increase the volume of the internal cavity of thechamber, thereby decreasing the total liquid volume of said inputcomposition present in said cavity during rotation of said centrifugalchamber per square inch of the internal surface area of the cavitycompared to the absence of gas in the chamber.

In some embodiments, the number of cells in the cavity during theincubation is at or about the number of the cells sufficient to form amonolayer on the internal surface of the cavity during rotation of thecentrifugal chamber at a force of at or about 2000 g and/or is no morethan 1.5 times or 2 times such a number of the cells.

In some of any such embodiments, the number of said cells in said inputcomposition is at or about the number of said cells sufficient to form amonolayer on the surface of said cavity during rotation of saidcentrifugal chamber at a force of at or about 2000 g at an internalsurface of the side wall; and/or the number of said cells in said inputcomposition is no more than 1.5 times or 2 times the number of saidcells sufficient to form a monolayer on the surface of said cavityduring rotation of said centrifugal chamber at a force of at or about2000 g at an internal surface of the side wall.

In some embodiments, the centrifugation is for a duration of between 120and 7200 seconds, such as between 120 and 3600 seconds, including valuesinclusive or within the range, such as whole-minute values inclusive orwithin the range.

In some embodiments, the methods include a) providing to an internalcavity of a centrifugal chamber that has an internal surface area of atleast at or about 1×10⁹ μm² or at least at or about 1×10¹⁰ μm²:i) aninput composition including cells and viral particles containing arecombinant viral vector, wherein: the number of cells in the inputcomposition is at least 1×10⁷ cells, and the viral particles are presentin the input composition at least at or about 1 infectious unit (IU) perone of said cells, and the input composition contains a liquid volumethat is less than the maximum volume of the internal cavity of thecentrifugal chamber; and ii) gas, at a volume that is up to theremainder of the maximum volume of the internal cavity of thecentrifugal chamber; and b) incubating the input composition, wherein atleast a portion of the incubation is carried out in said internal cavityof said centrifugal chamber while effecting rotation of said centrifugalchamber; and wherein the method generates an output compositioncontaining a plurality of the cells transduced with the viral vector.

In some embodiments, the number of cells is at least or about 50×10⁶cells; 100×10⁶ cells; or 200×10⁶ cells; and/or the viral particles arepresent at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8IU/cell, 3.2 IU/cell or 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.

In some of any such embodiments, the liquid volume of the inputcomposition is less than or equal to 200 mL, less than or equal to 100mL or less than or equal to 50 mL or less than or equal to 20 mL. Insome of any such embodiments, the volume of gas is up to 200 mL, up to180 mL, up to 140 mL or up to 100 mL.

In some of any such embodiments, said rotation is at a relativecentrifugal force at an internal surface of the side wall of the cavityor at a surface layer of the cells of at least at or about 1000 g, 1500g, 2000 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g or 3600 g.

In some embodiments, the methods are for large-scale processing.

In some embodiments, the composition in the cavity (e.g., inputcomposition) includes at least 50 mL, at least 100 mL, or at least 200mL, liquid volume, and/or at least or about 1 million cells per cm² ofthe internal surface area of the cavity during at least a portion ofsaid incubation.

In some embodiments, the maximum liquid volume of the input compositionpresent in the cavity at any one time during said incubation is no morethan at or about 2 times, 10 times, 25 times, 50 times, 100 times, 500times, or 1000 times the volume of a monolayer of said cells formed onthe inner surface of said cavity during rotation of said chamber, e.g.,at a force, e.g., effective force, of at or about 2000 g.

In some embodiments, the rotation of the chamber during at least aportion of the incubation is at a force of greater than at or about 200g, greater than at or about 300 g, or greater than at or about 500 g,such as greater than at or about 1000 g, 1500 g, 2000 g, 2500 g, 3000 g,or 3200 g, at an internal wall of the cavity of the centrifugal chamberand/or a layer, e.g., surface layer, of the cells. In some embodiments,the force is at least at or about 1000 g, 1500 g, 2000 g, or 2500 g,3000 g or 3200 g. In some embodiments, the force is at or about 2100 g,2200 g or 3000 g.

In some embodiments, the methods include incubating an input compositioncontaining cells and viral particles containing a recombinant viralvector, at least a portion of said incubating being carried out underrotating conditions, thereby generating an output composition containinga plurality of the cells transduced with the viral vector, wherein saidinput composition contains greater than or about 20 mL, 50 mL, at least100 mL, or at least 150 mL in volume, and/or said input compositioncomprises at least 1×10⁸ cells; and said rotating conditions comprise arelative centrifugal force on a surface layer of the cells of greaterthan about 1500 g.

In some embodiments of the methods, at least 25% or at least 50% of saidcells in the output composition are transduced with said viral vector;and/or at least 25% or at least 50% of said cells in the outputcomposition express a product of a heterologous nucleic acid containedwithin said viral vector.

In some of any such embodiments, said incubation is carried out in acavity of a centrifugal chamber and the number of said cells in saidinput composition is at or about the number of said cells sufficient toform a monolayer or a bilayer on the inner surface of said cavity duringsaid rotation.

In some embodiments, said centrifugal chamber includes an end wall, asubstantially rigid side wall extending from said end wall, and at leastone opening, wherein at least a portion of said side wall surrounds saidinternal cavity and said at least one opening is capable of permittingintake of liquid into said internal cavity and expression of liquid fromsaid cavity,

In some embodiments, said centrifugal chamber further includes a movablemember and said internal cavity is a cavity of variable volume definedby said end wall, said substantially rigid side wall, and said movablemember, said movable member being capable of moving within the chamberto vary the internal volume of the cavity.

In some of any such embodiments, the input composition in said cavitycontains a liquid volume of at least 20 mL or at least 50 mL and at orabout 1 million cells per cm² of the internal surface area of the cavityduring at least a portion of said incubation.

In some of any such embodiments, a further portion of the incubation iscarried out outside of the centrifugal chamber and/or without rotation,said further portion carried out subsequent to the at least a portioncarried out in the chamber and/or with rotation.

In some of any such embodiments, the at least a portion of theincubation carried out in the cavity of the centrifugal chamber and/orthe further portion of the incubation is effected at or at about 37°C.±2° C.

In some of any such embodiments, the incubation further includestransferring at least a plurality of the cells to a container duringsaid incubation and said further portion of the incubation is effectedin the container. In some embodiments, the transferring is performedwithin a closed system, wherein the centrifugal chamber and containerare integral to the closed system.

In some of any such embodiments, the incubation is carried out for atime between at or about 1 hour and at or about 96 hours, between at orabout 4 hours and at or about 72 hours, between at or about 8 hours andat or about 48 hours, between at or about 12 hours and at or about 36hours, between at or about 6 hours and at or about 24 hours, between ator about 36 hours and at or about 96 hours, inclusive; or the furtherportion of the incubation is carried out for a time between at or about1 hour and at or about 96 hours, between at or about 4 hours and at orabout 72 hours, between at or about 8 hours and at or about 48 hours,between at or about 12 hours and at or about 36 hours, between at orabout 6 hours and at or about 24 hours, between at or about 36 hours andat or about 96 hours, inclusive.

In some of any such embodiments, the incubation or further portion ofthe incubation is carried out for a time that is no more than 48 hours,no more than 36 hours or no more than 24 hours; or the further portionof the incubation is carried out for a time that is no more than 48hours, no more than 36 hours or no more than 24 hours.

In some of any such embodiments, the incubation is performed in thepresence of a stimulating agent; and/or the further portion of theincubation is performed in the presence of a stimulating agent.

In some of any such embodiments, the incubation is carried out for atime that is no more than 24 hours; the cells in the composition havenot been subjected to a temperature of greater than 30° C. for more than24 hours; and/or the incubation is not performed in the presence of astimulating agent.

In some of any such embodiments, the stimulating agent is an agentcapable of inducing proliferation of T cells, CD4+ T cells and/or CD8+ Tcells.

In some of any such embodiments, the stimulating agent is a cytokineselected from among IL-2, IL-15 and IL-7.

In some of any such embodiments, the output composition containingtransduced cells contains at least 1×10⁷ cells or at least 5×10⁷ cells.

In some of any such embodiments, the output composition containingtransduced cells contains at least 1×10⁸ cells, 2×10⁸ cells, 4×10⁸cells, 6×10⁸, 8×10⁸ cells or 1×10⁹ cells.

In some of any such embodiments, the cells are T cells. In someembodiments, the T cells are unfractionated T cells, isolated CD4+ Tcells and/or isolated CD8+ T cells.

In some of any such embodiments, the method results in integration ofthe viral vector into a host genome of one or more of the at least aplurality of cells and/or into a host genome of at least at or about 20%or at least at or about 30% or at least at or about 40% of the cells inthe output composition.

In some of any such embodiments, at least 2.5%, at least 5%, at least6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, or at least 75% of said cells in said inputcomposition are transduced with said viral vector by the method; and/orat least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 75% of said cells in said output composition are transduced withsaid viral vector; and/or at least 2.5%, at least 5%, at least 6%, atleast 8%, at least 10%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, or at least 75% of said cells in said outputcomposition express a product of a heterologous nucleic acid containedwithin said viral vector.

Particular embodiments include methods of transduction carried out byincubating an input composition comprising cells and viral vectorparticles under rotating conditions, whereby a plurality of the cellsare inoculated for transduction with the viral vector, wherein the inputcomposition includes a total volume greater than 50 mL, such as at least100 mL, or at least 150 mL in volume, and/or said input compositioncomprises at least 1×10⁸ cells; and the rotating conditions comprisecentrifugal force of greater than about 1500 g. In some suchembodiments, the incubation is carried out in a cavity of a centrifugalchamber and the number of said cells in said input composition is at orabout the number of said cells sufficient to form a monolayer on theinner surface of the cavity during the rotation. In some suchembodiments, at least 25% or at least 50% of said cells are transducedwith the viral vector.

In some embodiments, the methods result in at least 2.5%, at least 5%,at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, or at least 75% of said cells insaid input composition being transduced with the viral vector, and/orproduce an output composition in which at least 10%, at least 25%, atleast 30%, at least 40%, at least 50%, or at least 75% of the cells aretransduced with the vector and/or express a recombinant product encodedby the vector. In some embodiments, transduction efficiency is expressedfor a particular input amount or relative amount of virus. For example,in some embodiments, such efficiencies are achieved by the methods foran input composition comprising a virus at a ratio of about 1 or about 2IU per cells.

In some embodiments, among all the cells in said output compositionproduced by the methods, the average copy number of the recombinantviral vector is no more than about 10, no more than about 5, no morethan about 2.5, or no more than about 1.5. In some embodiments, amongthe cells in the output composition that contain the recombinant viralvector, the average copy number of the vector is no more than about 5,no more than about 2, no more than about 1.5, or no more than about 1.

In some of any such embodiments, among all the cells in said outputcomposition that contain the recombinant viral vector or into which theviral vector is integrated, the average copy number of said recombinantviral vector is no more than about 10, no more than about 5, no morethan about 2.5, or no more than about 1.5; or among the cells in theoutput composition, the average copy number of said vector is no morethan about 2, no more than about 1.5, or no more than about 1.

In some embodiments, the centrifugal chamber is integral to a closedsystem, for example, where the closed system includes the chamber and atleast one tubing line operably linked to the at least one opening via atleast one connector, such that liquid and gas are permitted to movebetween said cavity and said at least one tubing line in at least oneconfiguration of the system. The at least one tubing line typicallyincludes a series of tubing lines. The at least one connector typicallyincludes a plurality of connectors. The closed system may furtherinclude at least one container operably linked to the series of tubinglines, such that the at least one connection permits liquid and/or gasto pass between the at least one container and the at least one openingvia the series of tubing lines.

The at least one connector may include one or more connectors selectedfrom the group consisting of valves, luer ports, and spikes, e.g., arotational valve, such as a stopcock or multirotational port, and/or anaseptic connector.

The at least one container may include one or more bags, vials, and/orsyringes, and may include container(s) designated as a diluentcontainer, a waste container, a product collection container, outputcontainer, and/or an input container.

In some embodiments, the at least one container includes at least oneinput container including the virus and/or cells (which may be a singleinput container comprising the virus and cells or two input containerscomprising the virus and cells, respectively), a waste container, aproduct container, and at least one diluent or wash solution-containingcontainer, each connected to said cavity via said series of tubing linesand the at least one opening.

In some of any such embodiments, at least one container further includesa container that contains a gas prior to and/or during at least a pointduring said incubation and/or the closed system further includes amicrobial filter capable of taking in gas to the internal cavity of thecentrifugal chamber and/or the closed system contains a syringe port foreffecting intake of gas.

The methods in some embodiments further include, prior to and/or duringthe incubation, effecting intake of the input composition into saidcavity. The intake may include flow of liquid from the at least oneinput container into the cavity through said at least one opening. Theintake may include intake of virus from one input container and input ofcells from another, to produce the input composition for incubation.

In some embodiments, the method includes, prior to and/or during saidincubation, providing or effecting intake of gas into said cavity understerile conditions, said intake being effected by (a) flow of gas fromthe container that includes gas, (b) flow of gas from an environmentexternal to the closed system, via the microbial filter, or (c) flow ofgas from a syringe connected to the system at the syringe port.

In some embodiments, the effecting intake of the gas into the internalcavity of the centrifugal chamber is carried out simultaneously ortogether with the effecting intake of the input composition to theinternal cavity of the centrifugal chamber.

In some of any such embodiments, the input composition and gas arecombined in a single container under sterile conditions outside of thechamber prior to said intake of said input composition and gas into theinternal cavity of the centrifugal chamber.

In some embodiments, the effecting of the intake of the gas is carriedout separately, either simultaneously or sequentially, from theeffecting of the intake of the input composition into said cavity.

In some of any such embodiments, the intake of gas is effected bypermitting or causing flow of the gas from a sterile closed containercontaining the gas, an external environment through a microbial filter,or a syringe containing said gas.

In some of any such embodiments, the gas is air.

In some embodiments of the provided process methods, the incubation ispart of a continuous process, where the method further includes, duringat least a portion of the incubation, effecting continuous intake ofsaid input composition into the cavity, typically during rotation of thechamber, and during a portion of the incubation, effecting continuousexpression (i.e. outtake) of liquid from said cavity through said atleast one opening, typically during rotation of the chamber. Thecontinuous intake and outtake in some embodiments occur simultaneously.

In some embodiments, the method includes during a portion of saidincubation, effecting continuous intake of gas into said cavity duringrotation of the chamber; and/or during a portion of said incubation,effecting continuous expression of gas from said cavity.

In some embodiments, the method includes the expression of liquid andthe expression of gas from said cavity, where each is expressed,simultaneously or sequentially, into a different container.

In some of any such embodiments, at least a portion of the continuousintake and the continuous expression occur simultaneously.

In some embodiments, the incubation is part of a semi-continuousprocess, such as one in which the method further includes effectingintake of the input composition into the cavity through the at least oneopening, conducting all or part of the incubation, such as thecentrifugation, and then effecting expression of liquid from the cavity,and then repeating the process, whereby another input composition istaken in to the cavity, followed by centrifugation, followed byexpression. The process can be iterative and include several more roundsof intake, processing, and expression.

In some of any such embodiments, the incubation is part of asemi-continuous process, the method further including prior to saidincubation, effecting intake of said input composition, and optionallygas, into said cavity through said at least one opening; subsequent tosaid incubation, effecting expression of liquid and/or optionally gasfrom said cavity; effecting intake of another input compositionincluding cells and said viral particles containing a recombinant viralvector, and optionally gas, into said internal cavity; and incubatingsaid another input composition in said internal cavity, wherein themethod generates another output composition containing a plurality ofcells of the another input composition that are transduced with saidviral vector.

In some of any such embodiments, said providing or said intake of theinput composition into the cavity includes intake of a singlecomposition including the cells and the viral particles containing therecombinant viral vector; or intake of a composition including the cellsand a separate composition containing the viral particles containing therecombinant viral vector, whereby the compositions are mixed, effectingintake of the input composition.

The intake may include intake of a single composition containing thecells and the virus; or intake of a composition containing the cells anda separate composition containing the virus, whereby the compositionsare mixed, effecting intake of the input composition. In someembodiments of the continuous or semi-continuous process, at least 1×10⁸cells or at least 1×10⁹ cells or at least 1×10¹⁰ cells or more areprocessed in total, over the multiple rounds or continuous process.

In some embodiments, the method includes effecting rotation of thecentrifugal chamber prior to and/or during said incubation and effectingexpression of liquid from the cavity into said waste container followingthe incubation; effecting expression of liquid from the at least onediluent container into said cavity via the at least one opening andeffecting mixing of the contents of the cavity; and effecting expressionof liquid from said cavity into the product container, therebytransferring cells transduced with the viral vector into the productbag.

In some embodiments, the method further includes carrying out otherprocessing steps, or at least a portion of one or more other processingsteps, within the same chamber and/or closed system. In someembodiments, the one or more processing steps can includes processes inwhich the cells are isolated, such as separated or selected, stimulated,and formulated within the same chamber and/or closed system. In somecases, the one or more further processing steps also can include washingcells, suspending cells and/or diluting or concentrating cells, whichcan be carried out prior to or subsequent to any one or more of theprocessing steps for isolating, such as separating or selecting,stimulating, transducing and/or formulating the cells. In someembodiments, the one or more other processing steps can be carried outprior to, simultaneously with or subsequent to the incubation of cellswith the viral vector particles in the methods of transduction. In someembodiments, the one or more further processing steps, or a portion ofthe one or more further processing steps, can be carried out in a cavityof a centrifugal chamber that is the same or different as a cavity of acentrifugal chamber employed in the incubation of cells with the viralvector particles.

Among the provided processing methods, including isolation, e.g.selection, methods, stimulation methods, formulation methods and otherprocessing methods, are those carried out according to any of theembodiments as described above.

For example, in some embodiments, the method further includes (a)washing a biological sample (e.g., a whole blood sample, a buffy coatsample, a peripheral blood mononuclear cells (PBMC) sample, anunfractionated T cell sample, a lymphocyte sample, a white blood cellsample, an apheresis product, or a leukapheresis product) containingcells in a cavity of a chamber, prior to the incubation for isolating,e.g. selecting cells, and/or prior to the incubation for incubatingcells with viral vector particles, (b) isolating, e.g. selecting, thecells from a sample (e.g., a whole blood sample, a buffy coat sample, aperipheral blood mononuclear cells (PBMC) sample, an unfractionated Tcell sample, a lymphocyte sample, a white blood cell sample, anapheresis product, or a leukapheresis product) in a cavity prior to theincubation of such cells with viral vector particles and/or (c)stimulating cells in a cavity prior to and/or during the incubation ofsuch cells with viral vector particles, e.g., by exposing cells tostimulating conditions, thereby inducing cells of the input compositionto proliferate. In some embodiments, the isolating includesimmunoaffinity-based selection.

In some of any such embodiments, the method includes (a) washing abiological sample containing said cells in an internal cavity of acentrifugal chamber prior to said incubation; and/or (b) isolating saidcells from a biological sample, wherein at least a portion of theisolation step is performed in an internal cavity of a centrifugalchamber prior to said incubation; and/or (c) stimulating cells prior toand/or during said incubation, said stimulating including exposing saidcells to stimulating conditions, thereby inducing cells of the inputcomposition to proliferate, wherein at least a portion of the step ofstimulating cells is performed in an internal cavity of a centrifugalchamber.

In some embodiments, the methods may further include isolation, e.g.,selection, of the cells in the chamber, e.g., by immunoaffinity basedselection. In some embodiments, the isolation, e.g. selection, of cellsis carried out prior to the incubation of cells with the viral vectorparticles in the methods of transduction, whereby the isolated, such asselected, cells are the cells present in the input composition and/orincubated with the viral vector particles. In some embodiments, theisolation, e.g., selection, includes incubation of cells with aselection reagent, such as an immunoaffinity reagent. In someembodiments, at least a portion of the isolation, e.g. selection, step,such as incubation of cells with a selection reagent, e.g. animmunoaffinity reagent, is carried out in the cavity of a chamber,which, in some cases, can include rotation of the chamber, for example,for mixing of the reagent and cells.

In some embodiments, the methods may further include stimulating cellsprior to, during and/or subsequent to the incubation of cells with theviral vector particles, in which at least all or a portion of thestimulation can be carried out in a cavity of a centrifugal chamber. Insome embodiments, the stimulating conditions may include incubation ofcells in the presence of an agent capable of activating one or moreintracellular signaling domains of one or more components of a TCRcomplex, such as a primary agent that specifically binds to a member ofa TCR complex, e.g., CD3, and a secondary agent that specifically bindsto a T cell costimulatory molecule, e.g., CD28, CD137 (4-1-BB), OX40, orICOS, including antibodies such as those present on the surface of asolid support, such as a bead. In some embodiments, at least a portionof the stimulation, such as incubation of cells in the presence of astimulating condition, is carried out in the cavity of a chamber, which,in some cases, can include rotation of the chamber, for example, formixing of the reagent and cells.

In some of any such embodiments, the method includes formulating cells,such as cells produced or generated in accord with the provided methods,including cell transduced by the method, in a pharmaceuticallyacceptable buffer in an internal cavity of a centrifugal chamber,thereby producing a formulated composition. In some embodiments, themethods further include effecting expression of the formulatedcomposition to one or a plurality of containers. In some embodiments,the methods include the effecting of expression of the formulatedcomposition includes effecting expression of a number of the cellspresent in a single unit dose to one or each of said one or a pluralityof containers.

In some of any such embodiments, each of said a cavity of a centrifugalchamber is the same or different as a cavity of a centrifugal employedin one or more of the other steps and/or in the process of incubatingand/or rotating an input composition containing cells and viralparticles.

In some of any such embodiments, each of said centrifugal chambers isintegral to a closed system, said closed system including said chamberand at least one tubing line operably linked to the at least one openingvia at least one connector, whereby liquid and gas are permitted to movebetween said cavity and said at least one tubing line in at least oneconfiguration of said system.

The cells processed by the methods typically are primary cells, such ascells obtained from a subject, typically a human. The cells may bederived from a subject to which the therapy is to be administered, suchas one having a disease or condition targeted by a recombinant moleculeexpressed by a vector transduced, e.g., a recombinant antigen receptorsuch as a chimeric antigen receptor or transgenic TCR. Alternatively,the cells may be from a different subject. Thus, the methods encompassprocessing for autologous and allogeneic transfer. The cells may includesuspension cells, e.g., white blood cells, e.g., T cells, such asisolated CD8⁺ T cells, or isolated CD4⁺ T cells or subsets thereof, orNK cells.

In some embodiments, during the incubation, the centrifugal chamber isassociated with a sensor, for example, a sensor capable of monitoringthe position of the movable member, and control circuitry, such ascircuitry capable of receiving and transmitting information to and fromthe sensor, causing movement of said movable member, and/or that isfurther associated with a centrifuge and thus is capable of causingrotation of the chamber during said incubation.

In some embodiments, the chamber contains the movable member and duringthe incubation is located within a centrifuge and associated with asensor capable of monitoring the position of the movable member, andcontrol circuitry capable of receiving and transmitting information fromthe sensor and causing movement of the movable member, intake andexpression of liquid to and from said cavity via said one or more tubinglines, and rotation of the chamber via the centrifuge.

In some embodiments, the chamber, control circuitry, centrifuge, and/orsensor are housed within a cabinet, e.g., during the incubation.

In some embodiments of any of the viral transfer, e.g., transductionmethods, the recombinant viral vector encodes a recombinant receptor,which is thereby expressed by cells of the output composition. In someembodiments, the recombinant receptor is a recombinant antigen receptor,such as a functional non-T cell receptor, e.g., a chimeric antigenreceptor (CAR), or a transgenic T cell receptor (TCR). In someembodiments, the recombinant receptor is a chimeric receptor containingan extracellular portion that specifically binds to a ligand and anintracellular signaling portion containing an activating domain and acostimulatory domain.

In some of any such embodiments, the cells include primary human T cellsobtained from a human subject and prior to the incubation with viralvector particles and/or prior to completion of the transduction and/or,where the method includes formulation, prior to the formulation, theprimary human T cells have not been present externally to the subject ata temperature of greater than 30° C. for greater than 1 hour, greaterthan 6 hours, greater than 24 hours, or greater than 48 hours or priorto the incubation and/or prior to the completion of the transduction,and/or where the method includes formulation, prior to the formulation,the primary human T cells have not been incubated in the presence of anantibody specific for CD3 and/or an antibody specific for CD28 and/or acytokine, for greater than 1 hour, greater than 6 hours, greater than 24hours, or greater than 48 hours.

Provided herein are methods for isolation, e.g. selection, of cellsincluding (a) incubating a selection reagent and primary cells in aninternal cavity of a centrifugal chamber under mixing conditions,whereby a plurality of the primary cells bind to said selection reagentand (b) separating the plurality of the primary cells from another oneor more of the primary cells based on binding to the selection reagent,thereby enriching the primary cells based on binding to the selectionreagent, wherein the centrifugal chamber is rotatable around an axis ofrotation and the internal cavity has a maximum volume of at least 50 mL,at least 100 mL, or at least 200 mL. In some embodiments, the methodsfor isolation, e.g. selection, occur in a closed system. In someembodiments, prior to the step of separating the plurality of cells, thecells incubated with the selection reagent, are expressed from ortransferred out of the chamber, but maintained in the closed system. Insome embodiments, optionally, subsequent to incubation with theselection reagent and prior to separating the cells, the method furtherincludes one or more washing steps, which in some cases, can beperformed in the cavity of the chamber in accord with the providedmethods. In some embodiments, the step of separating the cells can beeffecting using a solid support, such as using an immunoaffinity-column,including those for magnetic separation, which can be contained in theclosed system.

Provided herein are methods for stimulation of cells, includingincubating a stimulation agent and primary cells under conditionswhereby the stimulation agent binds to a molecule expressed by aplurality of the primary cells and the plurality of the cells areactivated or stimulated, wherein at least a portion of the incubation iscarried out in an internal cavity of a centrifugal chamber under mixingconditions, where the centrifugal chamber is rotatable around an axis ofrotation and the internal cavity has a maximum volume of at least 50 mL,at least 100 mL, or at least 200 mL.

In some embodiments, the methods of stimulation are performed as part ofa process that includes transducing cells, whereby all or a part of suchprocess is performed in a centrifugal chamber and/or as part of the sameclosed system. In some embodiments, the primary cells that arestimulated with a stimulation agent include or are cells obtainedfollowing isolation, e.g. selection, of cells from a biological sample,such as in accord with the provided methods. In some embodiments, atleast a portion of the stimulation is carried out simultaneously orduring the incubation of cells with the viral vector particles, suchthat the primary cells include or are cells present in the inputcomposition and/or are cells in which transduction has occurred or isinitiated. In some embodiments, at least a portion of the stimulation iscarried out prior to the incubation of cells with the viral vectorparticles, such that the cells incubated with the viral vector particlesare stimulated cells, which, in some cases, includes proliferatingcells.

In some embodiments, at least a portion of the one of more otherprocessing steps of the method, including isolation, e.g. selection,stimulation, washing and/or formulation, that is carried out in achamber includes where the chamber includes an end wall, a substantiallyrigid side wall extending from said end wall, and at least one opening,wherein at least a portion of the side wall surrounds the internalcavity and the at least one opening is capable of permitting intake ofliquid into the internal cavity and expression of liquid from thecavity.

Provided herein are compositions containing transduced cells produced bythe methods of any of the above embodiments. In some of any suchembodiments, the composition contains cells that are primary cellsand/or human cells and/or include white blood cells, and/or T cells,and/or NK cells. In some of any such embodiments, the compositioncontains at least 5×10⁷ cells, 1×10⁸ cells, 2×10⁸ cells, 4×10⁸ cells,6×10⁸ cells, 8×10⁸ cells or 1×10⁹ cells. In some of any suchembodiments, the composition contains a therapeutically effective numberof cells for use in adoptive T cell therapy. In some of any suchembodiments, the cells are T cells and subsequent to transduction, thecells in the composition are not subjected to cell expansion in thepresence of a stimulating agent and/or the cells are not incubated at atemperature greater than 30° C. for more than 24 hours or thecomposition does not contain a cytokine or the composition does notcontain a stimulating agent that specifically binds to CD3 or a TCRcomplex.

Provided herein are compositions containing at least 1×10⁷ or at least5×10⁷ T cells, at least a plurality of which are transduced with arecombinant viral vector, where subsequent to transduction, the cells inthe composition have not been subjected to cell expansion in thepresence of a stimulating agent and/or the cells have not been incubatedat a temperature greater than 30° C. for more than 24 hours and/or atleast 30, 40, 50, 60, 70, or 80% of the T cells in the compositioncontain high surface expression of CD69 or TGF-beta-II. In someembodiments, the composition contains at least 1×10⁸ cells, 2×10⁸ cells,4×10⁸ cells, 6×10⁸, 8×10⁸ cells or 1×10⁹ cells.

In some of any such embodiments, the T cells are unfractionated T cells,isolated CD8+ T cells, or isolated CD4+ T cells.

In some of any such embodiments, at least 2.5%, at least 5%, at least6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, or at least 75% of said cells in saidcomposition are transduced with the viral vector.

In some of any such embodiments, the viral vector encodes a recombinantreceptor and transduced cells in the composition express the recombinantreceptor. In some embodiments, the recombinant receptor is a recombinantantigen receptor. In some embodiments, the recombinant antigen receptoris a functional non-T cell receptor. In some embodiments, the functionalnon-T cell receptor is a chimeric antigen receptor (CAR). In someembodiments, the recombinant receptor is a chimeric receptor containingan extracellular portion that specifically binds to a ligand and anintracellular signaling portion containing an activating domain and acostimulatory domain. In some embodiments, the recombinant antigenreceptor is a transgenic T cell receptor (TCR).

In some of any such embodiments, among all the cells in the composition,the average copy number of the recombinant viral vector is no more thanabout 10, no more than 8, no more than 6, no more than 4, or no morethan about 2, or among the cells in the composition transduced with therecombinant viral vector, the average copy number of said vector is nomore than about 10, no more than 8, no more than 6, no more than 4, orno more than about 2.

In some of any such embodiments, the composition contains apharmaceutically acceptable excipient.

Provided herein are methods of treatment, including administering to asubject having a disease or condition the composition of any of theabove embodiments. In some embodiments, the transduced T cells in thecomposition exhibit increased or longer expansion and/or persistence inthe subject than transduced T cells in a composition in which,subsequent to transduction, the cells in the composition have beensubjected to cell expansion in the presence of a stimulating agentand/or the cells have been incubated at a temperature greater than 30°C. for more than 24 hours.

In some of any such embodiments, the recombinant receptor, chimericantigen receptor or transgenic TCR specifically binds to an antigenassociated with the disease or condition. In some embodiments, thedisease or condition is a cancer, an autoimmune disease or disorder, oran infectious disease.

Provided herein are compositions containing at least 1×10⁷ cells and atleast at or about 1 infectious unit (IU) per cell of viral particlescontaining a recombinant viral vector. In some embodiments, the cellscontain at least or about 50×10⁶ cells, 100×10⁶ cells, or 200×10⁶ cells,and/or said viral particles are present in the composition in an amountthat is at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.

In any of such embodiments, the liquid volume of the composition is lessthan or equal to 220 mL, less than or equal to 200 mL, less than orequal to 100 mL, less than or equal to 50 mL or less than or equal to 20mL.

In some of any such embodiments, the cells are primary cells. In some ofany such embodiments, the cells are human cells. In some of any suchembodiments, the cells include suspension cells, the cells include whiteblood cells and/or the cells include T cells or NK cells. In someembodiments, the cells are T cells and the T cells are unfractionated Tcells, isolated CD8+ T cells, or isolated CD4+ T cells.

In some of any such embodiments, the viral vector encodes a recombinantreceptor. In some embodiments, the recombinant receptor is a recombinantantigen receptor. In some embodiments, the recombinant antigen receptoris a functional non-T cell receptor. In some embodiments, the functionalnon-T cell receptor is a chimeric antigen receptor (CAR). In someembodiments, the recombinant receptor is a chimeric receptor containingan extracellular portion that specifically binds to a ligand and anintracellular signaling portion containing an activating domain and acostimulatory domain. In some embodiments, the recombinant antigenreceptor is a transgenic T cell receptor (TCR).

Provided herein are centrifugal chambers rotatable around an axis ofrotation, including an internal cavity containing the composition of anyof the above embodiments.

Provided herein are centrifugal chambers rotatable around an axis ofrotation, containing an internal cavity containing (a) a compositioncontaining at least 5×10⁷ primary T cells transduced with a recombinantviral vector and/or (b) a composition containing at least 5×10⁷ primaryT cells and viral particles containing a recombinant viral vector.

In some of any such embodiments, the chamber further contains an endwall, a substantially rigid side wall extending from said end wall, andat least one opening, wherein at least a portion of said side wallsurrounds said internal cavity and said at least one opening is capableof permitting intake of liquid into said internal cavity and expressionof liquid from said cavity.

In some of any such embodiments, said composition in said cavitycontains at least 1×10⁸ cells, 2×10⁸ cells, 4×10⁸ cells, 6×10⁸ cells,8×10⁸ cells or 1×10⁹ of the cells.

In some of any such embodiments, the T cells are unfractionated T cells,isolated CD8+ T cells, or isolated CD4+ T cells.

In some of any such embodiments of the chamber, at least 2.5%, at least5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, or at least 75% of said cellsin said composition are transduced with a viral vector.

In some of any such embodiments of the chamber, the viral vector encodesa recombinant receptor and cells in the composition express therecombinant receptor. In some embodiments, the recombinant receptor is arecombinant antigen receptor. In some embodiments, the recombinantantigen receptor is a functional non-T cell receptor. In someembodiments, the functional non-T cell receptor is a chimeric antigenreceptor (CAR). In some embodiments, the recombinant receptor is achimeric receptor containing an extracellular portion that specificallybinds to a ligand and an intracellular signaling portion containing anactivating domain and a costimulatory domain. In some embodiments, therecombinant antigen receptor is a transgenic T cell receptor (TCR).

In some of any such embodiments of the chamber, among all the cells inthe composition, the average copy number of said recombinant viralvector is no more than about 10, no more than 8, no more than 6, no morethan 4, or no more than about 2 or among the cells in the compositiontransduced with the recombinant viral vector, the average copy number ofsaid vector is no more than about 10, no more than 8, no more than 6, nomore than 4, or no more than about 2.

Provided herein are centrifugal chambers rotatable around an axis ofrotation, including an internal cavity containing the composition of anyof the above embodiments. In some embodiments, the chamber furthercontains a volume of gas up to the maximum volume of the internal cavityof the chamber. In some embodiments, the gas is air.

In some of any such embodiments of the chamber, the chamber is rotatablearound an axis of rotation and includes an end wall, a substantiallyrigid side wall extending from said end wall, and at least one opening,wherein at least a portion of said side wall surrounds said internalcavity and said at least one opening is capable of permitting intake ofliquid into said internal cavity and expression of liquid from saidcavity. In some embodiments the side wall is curvilinear. In someembodiments the side wall is generally cylindrical.

In some of any such embodiments of the chamber, said at least oneopening includes an inlet and an outlet, respectively capable ofpermitting said intake and expression or said at least one openingincludes a single inlet/outlet, capable of permitting said intake andsaid expression. In some of any such embodiments of the chamber, aid atleast one opening is coaxial with the chamber and is located in the endwall.

In some of any such embodiments, the chamber further includes a movablemember and said internal cavity is a cavity of variable volume definedby said end wall, said substantially rigid side wall, and said movablemember, said movable member being capable of moving within the chamberto vary the internal volume of the cavity. In some embodiments, themovable member is a piston and/or the movable member is capable ofaxially moving within the chamber to vary the internal volume of thecavity.

In some of any such embodiments, the internal surface area of saidcavity is at least at or about 1×10⁹ μm², the internal surface area ofsaid cavity is at least at or about 1×10¹⁰ μm², the length of said rigidwall in the direction extending from said end wall is at least about 5cm, the length of said rigid wall in the direction extending from saidend wall is at least about 8 cm and/or the cavity contains a radius ofat least about 2 cm at least one cross-section.

In some of any such embodiments of the chamber, the liquid volume ofsaid composition present in said cavity is between or between about 0.5mL per square inch of the internal surface area of the cavity (mL/sq.in)and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5 mL/sq.in. and 1mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and 2.5 mL/sq.in. or2.5 mL/sq.in. and 5 mL/sq.in. In some of any such embodiments, theliquid volume of said composition present in said cavity is at least 0.5mL/sq.in., 1 mL/sq.in., 2.5 mL/sq.in., or 5 mL/sq.in.

Provided herein are closed systems containing the centrifugal chamber ofany of the above embodiments. In some of any such embodiments of theclosed system, the centrifugal chamber is capable of rotation at a speedup to 8000 g, wherein the centrifugal chamber is capable of withstandinga force of 500, 1000, 1500, 2000, 2500, 3000 or 3200 g, withoutsubstantially yielding, bending, or breaking or otherwise resulting indamage of the chamber and/or while substantially holding a generallycylindrical shape under such force.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows transduction efficiency calculated as percentage of CD3⁺ Tcells with surface expression of a chimeric antigen receptor (CAR)encoded by a viral vector, following incubation under various conditionsas described in Example 1. FIG. 1B shows population doublings over asix-day period during the transduction study described in Example 1.

FIG. 2 shows transduction efficiency calculated as percentage of CD3⁺ TCells with surface expression of a CAR encoded by a viral vectorfollowing incubation under the indicated conditions as described inExample 2.

FIG. 3 shows transduction efficiency calculated as percentage of CD3⁺ TCells with surface expression of a CAR encoded by a viral vectorfollowing incubation under various conditions as described in Example 3.

FIG. 4 shows mean vector copy number (VCN) of a viral vector inindicated cell populations following transduction under variousconditions as described in Example 4.

FIG. 5 provides a schematic representation of an embodiment of a closedsystem (processing kit) for use in embodiments of the provided methods.The depicted exemplary system includes a generally cylindricalcentrifugal chamber (1), rotatable around an axis of rotation andincluding an end wall (13), a rigid side wall (14), and a piston (2),defining an internal cavity (7) of the chamber. The chamber furtherincludes an inlet/outlet opening (6) to permit flow of liquid and gas inand out of the cavity in at least some configurations of the system. Theopening (6) is operably linked with a series of tubing lines (3) andconnectors, including stopcock valves (4) and various additionalcontainers. Clamps (5) are also depicted.

FIG. 6A shows population doublings over a ten-day period during thestudy described in Example 6. FIG. 6B shows percent viability of cellsover a ten-day period during the study described in Example 6.

FIG. 7 provides a schematic representation of an embodiment of a closedsystem (processing kit) for use in embodiments of the provided methods.The depicted exemplary system includes a generally cylindricalcentrifugal chamber (1), rotatable around an axis of rotation andincluding an end wall (13), a rigid side wall (14), and a piston (2),defining an internal cavity (7) of the chamber. The chamber furtherincludes an inlet/outlet opening (6) to permit flow of liquid and gas inand out of the cavity in at least some configurations of the system. Theopening (6) is operably linked with a series of tubing lines (3) andconnectors, including stopcock valves (4), various additionalcontainers, and an air filter (15) coupled to a removable cap (16).Clamps (5) are also depicted.

FIG. 8A shows transduction efficiency calculated as percentage of CD3⁺ TCells with surface expression of a CAR encoded by a viral vectorfollowing incubation under the indicated conditions as described inExample 8A. FIG. 8B shows transduction efficiency calculated aspercentage of CD3⁺ T Cells with surface expression of a CAR encoded by aviral vector following incubation under the indicated conditions asdescribed in Example 8B. FIG. 8C shows mean vector copy number (VCN) ofa viral vector in indicated cell populations following transductionunder various conditions as described in Example 8B.

FIG. 9A shows transduction efficiency calculated as percentage of CD3⁺ TCells with surface expression of a CAR encoded by a viral vectorfollowing incubation under the indicated conditions as described inExample 9. FIG. 9B shows mean vector copy number (VCN) of a viral vectorin indicated cell populations following transduction under variousconditions as described in Example 9.

FIG. 10 shows transduction efficiency calculated as percentage of CD3⁺ TCells with surface expression of a CAR encoded by a viral vectorfollowing incubation under the indicated conditions as described inExample 10.

FIG. 11 provides a schematic representation of an embodiment of a closedsystem (processing kit) for use in embodiments of the provided methods.The depicted exemplary system includes a generally cylindricalcentrifugal chamber (1), rotatable around an axis of rotation andincluding an end wall (13), a rigid side wall (14), and a piston (2),defining an internal cavity (7) of the chamber. The chamber furtherincludes an inlet/outlet opening (6) to permit flow of liquid and gas inand out of the cavity in at least some configurations of the system. Theopening (6) is operably linked with a series of tubing lines (3) andconnectors, including stopcock valves (4) and ports (18), and variousadditional containers, including a plurality of output bags (17). Clamps(5) are also depicted.

DETAILED DESCRIPTION

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. Methods of Cell Processing and Associated Systems, Kits, and Devices

Provided are methods for processing cells, for example, to generatecompositions of cells for use in adoptive cell therapy. The methodsinclude those for transferring recombinant viral vectors to the cells,such as by viral transduction. The viral vectors generally encoderecombinant molecules to be expressed in the cells, e.g., for use incell therapy. Processing steps of the methods can also or alternativelyinclude all or a portion of cell washing, dilution, selection,isolation, separation, cultivation, stimulation, packaging, and/orformulation. The methods generally allow for the processing, e.g.,selection or separation and/or transduction, of cells on a large scale(such as in compositions of volumes greater than at or about 50 mL). Oneor more of the cell processing steps generally are carried out in theinternal cavity of a centrifugal chamber, such as a substantially rigidchamber that is generally cylindrical in shape and rotatable around anaxis of rotation, which can provide certain advantages compared to otheravailable methods. In some embodiments, all processing steps are carriedout in the same centrifugal chamber. In some embodiments, one or moreprocessing steps are carried out in different centrifugal chambers, suchas multiple centrifugal chambers of the same type.

The provided methods offer various advantages compared with availablemethods for cell processing, including for transduction and selection,particularly those for large-scale cell processing. Certain availablemethods have not been entirely satisfactory, for example, due to lessthan optimal efficacy, accuracy, reproducibility, cost and timeexpenditure, risk of error, complexity, and need for user handling andbiosafety facilities. In some embodiments, the provided methods aresuitable for large-scale and/or clinical-grade cell production, whilestill providing desirable features otherwise available only withsmall-scale production methods, and offering additional advantages notprovided by available methods. For example, the methods for celltransduction and/or affinity-based selection offer advantages comparedwith available methods performed in flexible plastic bags or plasticmulti-well plates.

In some embodiments, the centrifugal chamber and/or its internal cavityin which the cells are processed is surrounded or defined at least inpart by rigid or substantially rigid material. Incubation in a cavitybound by such materials, such as hard plastic, permits centrifugationunder certain conditions, such as forces higher than those that may beused with bags used in other large-scale cell processing methods. Forexample, in some embodiments, the chamber and cavity withstandcentrifugation at a force, e.g., a relative centrifugal force, of leastat or about 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g, asmeasured for example at an internal or external wall of the chamber orcavity, or at one or more cell, such as layer of cells, withoutsubstantially yielding, bending, or breaking or otherwise resulting indamage of the chamber or cavity holding the cells, such that the chamberand/or cavity substantially hold their shape under such force.

Accordingly, the chamber and/or its internal cavity typically aresurrounded by all or a portion of a rigid or semi-rigid side wall, suchas one made of hard plastic, which holds its shape under the centrifugalforce applied. The side wall generally is curvilinear, e.g., cylindricalor generally cylindrical, and typically extends from one or two endwalls of the chamber, the internal side of one or both of which may alsodefine the boundaries of the internal cavity. The end walls in someembodiments are also made of rigid materials, and in some embodimentsmay include more flexible materials. In some embodiments, while a wallis made of rigid material or substantially rigid material, it maynonetheless be lined and/or coated with flexible material and/or containsmall portions which are more flexible, so long as the cavity as a wholemaintains its overall shape during the conditions of the methods.

The centrifugal chamber generally is rotatable around an axis ofrotation, and the cavity typically is coaxial with the chamber. In someembodiments, the centrifugal chamber further includes a movable member,such as a piston, which generally is capable of movement (e.g., axialmovement) within the chamber, to vary the volume of the cavity. Thus, inparticular embodiments, the internal cavity is bound by the side walland end wall of the chamber and the movable member, and has a variablevolume that may be adjusted by moving the movable member. The movablemember may be made of rigid, substantially or generally rigid, flexiblematerials, or combinations thereof.

The chamber generally also includes one or more opening(s), such as oneor more inlet, one or more outlet, and/or one or more inlet/outlet,which can permit intake and expression of liquid and/or gas to and fromthe cavity. In some cases, the opening can be an inlet/outlet where bothintake and expression of the liquid and/or gas occurs. In some cases,the one or more inlets can be separate or different from the one or moreoutlets. The opening or openings may be in one of the end walls. In someembodiments, liquid and/or gas is taken into and/or expressed from thecavity by movement of the movable member to increase and/or decrease thecavity's volume. In other embodiments, liquid and/or gas may be takeninto and/or expressed from the cavity through a tubing line or otherchannel that is or is placed in connection with the opening, forexample, by placing the line or channel in connection with and controlof a pump, syringe, or other machinery, which may be controlled in anautomated fashion.

In some embodiments, the chamber is part of a closed system, such as asterile system, having various additional components such as tubinglines and connectors and caps, within which processing steps occur.Thus, in some embodiments, the provided methods and/or steps thereof arecarried out in a completely closed or semi-closed environment, such as aclosed or semi-closed sterile system, facilitating the production ofcells for therapeutic administration to subjects without the need for aseparate sterile environment, such as a biosafety cabinet or room. Themethods in some embodiments are carried out in an automated or partiallyautomated fashion.

In some embodiments, the chamber is associated with a centrifuge, whichis capable of effecting rotation of the chamber, such as around its axisof rotation. Rotation may occur before, during, and/or after theincubation in one or more of the processing steps. Thus, in someembodiments, one or more of the various processing steps is carried outunder rotation, e.g., at a particular force. The chamber is typicallycapable of vertical or generally vertical rotation, such that thechamber sits vertically during centrifugation and the side wall and axisare vertical or generally vertical, with the end wall(s) horizontal orgenerally horizontal. One exemplary chamber is depicted within exemplaryclosed systems depicted in FIG. 5, FIG. 7 or FIG. 11.

The processing steps of the methods (e.g., the steps carried out inwhole or in part in the chamber) may include any one or more of a numberof cell processing steps, alone or in combination. In particularembodiments, the processing steps include transduction of the cells withviral vector particles containing a retroviral vector, such as oneencoding a recombinant product for expression in the cells, where atleast a part of the incubation with the viral vector particles isperformed in the chamber to initiate transduction. The methods mayfurther and/or alternatively include other processing steps, such assteps for the isolation, separation, selection, cultivation (e.g.,stimulation of the cells, for example, to induce their proliferationand/or activation), washing, suspension, dilution, concentration, and/orformulation of the cells. In some embodiments, the method includesprocessing steps carried out in an order in which: cells, e.g. primarycells, are first isolated, such as selected or separated, from abiological sample; resulting isolated or selected cells are stimulatedin the presence of a stimulation reagent; stimulated cells are incubatedwith viral vector particles for transduction; and transduced cells areformulated in a composition. In some embodiments, the stimulation isadditionally or alternatively performed during at least a part of theincubation with the viral vector particles. In some cases, stimulationis additionally or alternatively carried out after incubation of cellswith the viral vector particles. In some cases, the methods do notinclude a step of stimulating the cells. In some embodiments, the methodcan include one or more processing steps from among washing, suspending,diluting and/or concentrating cells, which can occur prior to, during orsimultaneous with or subsequent to one or more of the isolation, such asseparation or selection, stimulation, transduction and/or formulationsteps. All or a portion of each of the processing steps may be performedin a closed system, such as in a centrifugal chamber. In aspects of themethods, the processes need not be performed in the same closed system,such as in the same centrifugal chamber, but can be performed under adifferent closed system, such as in a different centrifugal chamber; insome embodiments, such different centrifugal chambers are at therespective points in the methods placed in association with the samesystem, such as placed in association with the same centrifuge. In someembodiments, all processing steps are performed in a closed system, inwhich all or a portion of each one or more processing step is performedin the same or a different centrifugal chamber.

In some embodiments, the methods provide the ability to transduce thecells at a higher transduction efficiency compared with availablemethods, e.g., by carrying out all or a part of transduction at highercentrifugal forces/speeds, and/or by allowing easy, automated, and/orindependent control or adjustment of various parameters, such as volumeor amount of reagents, speed, and/or temperature. In some embodiments,the methods increase efficacy and/or reduce variability (increasingreproducibility), e.g., by streamlining and/or decreasing the number ofuser-interactions and/or handling steps, such as by providing automatedor semi-automated control of the various steps.

In some embodiments, by virtue of carrying out one or more, e.g., all ora portion of all, of the processing steps within a closed system, suchas a sterile closed system, the provided methods allow for thelarge-scale preparation of cells for clinical use without exposing thecells to non-sterile conditions and without the use of a separatesterile room or cabinet. In some embodiments, the cells are isolated,separated or selected, stimulated, transduced, washed, and formulatedwithin the closed system, e.g., in an automated fashion. In someembodiments, the methods are advantageous in that they are streamlined,e.g., require fewer steps, less user handling or intervention, e.g., bybeing carried out in a single, closed system and/or in an automatedfashion. For example, in some embodiments, the methods provideimprovement over methods for processing cells for use in clinicalapplications, which may require transduction in bags in a centrifuge orplate, by mixing viral vector particles and cells at appropriate ratiosin a biosafety cabinet, followed by transportation of the plate or bagto the centrifuge for transduction or other processing step, andadditional steps that may also require handling. In some embodiments,the provided methods are less manual and/or labor-intensive compared tosuch available methods, requiring a reduced degree or quantity ofhandling and user interaction.

In some embodiments, the methods allow for a greater degree of processcontrol compared with available methods. For example, the methods insome embodiments allow for the independent control of variousparameters, e.g., in an automated fashion. For example, the methods mayallow independent control of volume, amount, and/or concentration ofvarious components and reagents used in and processed with the methodsor various conditions used in one or more of the processes or methods.They generally permit control of the duration of one or more varioussteps of the methods, and/or the control of the ratio of cells in aparticular incubation or composition, liquid volume, and/or surface areaof the vessel being used for the processing, such as the chamber orcavity. The ability to control such parameters independently,particularly in an automated fashion and independently of one another,can allow a user to easily optimize and carry out the methods forindividual conditions.

Also provided are systems, devices, and apparatuses for use with suchmethods, kits containing the same, and methods of use of thecompositions and cells produced by the methods. For example, providedare methods of treatment and therapeutic use of the cells andcompositions produced by the methods, such as in adoptive cell therapy.Also provided are pharmaceutical compositions and formulations for usein such therapies.

II. Centrifugal Chambers and Associated Systems and Devices

In some embodiments, all or part of one or more of the processing steps,such as the incubation with virus to initiate or effect transductionand/or incubation with beads for immunoaffinity-based separation and/orone or more other processing steps as described, is carried out in acentrifugal chamber. In particular, such steps and incubations generallyare carried out in an internal cavity of such a chamber, which can be asame or different centrifugal chamber for each of the one or moreprocesses.

The centrifugal chamber is generally capable of being rotated, e.g., bya centrifuge that may be associated with the chamber during theincubation. In some embodiments, the centrifuge chamber is rotatablearound an axis of rotation, such as a vertical or generally orsubstantially vertical axis of rotation. In some embodiments, thecentrifuge chamber includes an end wall and a side wall, at least aportion of which surrounds or encircles the internal cavity of thechamber. The centrifuge chamber generally also includes another endwall, from which the side wall extends in the opposite direction.

The internal cavity generally is bound on its outside by the internalsides of all or a portion of the end wall, all or a portion of the sidewall, and all or a portion of another end wall of the chamber or anothersurface or object, such as a movable member within the chamber, such asa piston. The cavity in some aspects is hollow. In other aspects, asolid or hollow object is contained within part of the internal space ofthe cavity, such as a tube or channel.

In some aspects, the cavity is of variable volume, meaning that thetotal volume available within the cavity that may be occupied, e.g., byliquid or gas, may be varied, for example, by movement of the moveablemember, e.g., a piston. In some embodiments, such movement is possibleduring various steps of the methods, such as during the incubation toinitiate or effect the transduction or selection or steps subsequentand/or prior thereto. The movement in some embodiments may be effectedin an automated fashion, such as by a pre-specified program run byvirtue of circuitry and machinery associated with the chamber, such assensors and motors sensing and controlling position of the movablemember and other aspects of the process and circuitry for communicatingbetween the sensors and one or more components.

The side wall of the chamber, or the portion thereof that surrounds theinternal cavity of the chamber (and thus the shape of the cavity),typically is curvilinear, such as cylindrical, substantiallycylindrical, or generally cylindrical. The term cylindrical is generallyunderstood to those in the art to refer to a particular type ofcurvilinear surface, formed by the points at a fixed distance from agiven line segment, deemed the axis of a cylindrical shape. “Generallycylindrical” refers to a shape or surface having a configuration that isapproximately cylindrical in shape or structure, such as one thatappears cylindrical to the eye or is nearly cylindrical, but allows forsome degree of variability. For example, the term encompasses shapes andsurfaces of which not every point is at the same distance from the axis,and permits some degree of contouring and/or tapering, so long as theshape or surface appears cylindrical and/or has a primarily cylindricalshape. It also encompasses shapes in which the majority of the shape iscylindrical, such as where the majority of an outer wall of thecentrifuge chamber is cylindrical or substantially cylindrical in shapebut relatively minor portions of it adopt another configuration, forexample, tapering or contouring at or approaching one or more ends ofthe wall. In some embodiments, the portion of the side wall of thechamber that surrounds the cavity is cylindrical, whereas other portionsof the wall may not be cylindrical.

In some embodiments, all or portions of the chamber and/or cavity arerigid or substantially rigid. For example, all or part of the side wallmay be rigid or substantially rigid, for example to allow the chamberand cavity to withstand force, e.g., as applied during centrifugation athigh speeds, for example, at a force (relative centrifugal force (RCF))at the internal surface of the side wall of the cavity and/or at asurface layer of the cells of greater than at or about 200 g, greaterthan at or about 300 g, or greater than at or about 500 g, such asgreater than at or about 600 g, 800 g, 1100 g, 1000 g, 1500 g, 1600 g,2000 g, 2200 g, 2500 g, 3000 g or 3200 g; or at least at or about 600 g,800 g, 1000 g, 1100 g, 1500 g, 1600 g 2000 g, 2200 g, 2500 g, 3000 g, or3200 g, such as at or about 2100 g or 2200 g. In some embodiments, theRCF at the internal surface of the side wall of the cavity and/or at asurface layer of the cells is greater than at or about or is at or about1100 g, 1200 g, 1400 g, 1600 g, 1800 g, 2000 g, 2200 g or more. Incontrast, available methods for processing cells on a large scale, e.g.,greater than 50 or 100 mL volume, using flexible bags, may only permitcentrifugation at a relative centrifugal force of no more than 200 g,500 g, or 1000 g. Thus, the provided methods can produce greaterefficacy compared to such methods.

The term “relative centrifugal force” or RCF is generally understood tobe the effective force imparted on an object or substance (such as acell, sample, or pellet and/or a point in the chamber or other containerbeing rotated), relative to the earth's gravitational force, at aparticular point in space as compared to the axis of rotation. The valuemay be determined using well-known formulas, taking into account thegravitational force, rotation speed and the radius of rotation (distancefrom the axis of rotation and the object, substance, or particle atwhich RCF is being measured).

The object, particle, or location (or average thereof) at which RCF isexpressed or determined in a given case may be specified. For example,an RCF value or approximate value or range in some context herein isgiven for a particular portion or location within the centrifugalchamber used in such methods, such as at the internal surface of theside wall of the chamber's cavity in which the cells are processed, suchas at any point along the surface of the cylindrical side wall of thecavity or at the average radial distance thereof. Similarly, the RCFvalue may be given for a radial distance or average radial distancewithin another container, such as a bag, in which cells are processed,relative to the axis of rotation. In other embodiments, the RCF is givenfor the location of the sample or composition as a whole or at one ormore particular cells or average or layer thereof, during the rotation.For example, the value may be the RCF at a surface layer of the cells inthe chamber or other container during rotation, such as at the cellsurface at the interface between a liquid in which the cells are beingspun and the cells themselves.

In general, the RCF is calculated by the formula 1.119×10⁻⁵ (rpm)²r (or1.12×10−5×(rpm)2*r), where r=the radius (i.e., the distance in cm of agiven particle, object or substance from the axis of rotation),rpm=revolutions per minute. For example, in some embodiments, the RCF atthe internal surface of the side wall of internal processing cavity inwhich cells are processed may be calculated using this formula, in whichr is the distance between a point on the internal surface of the sidewall and the axis of rotation. Alternatively, the RCF at a cell orsurface layer of cells (such as the interface between the cell layer(s)and liquid during rotation) may be calculated using the formula, inwhich r is the distance between the cell, surface layer, and/orinterface, or an average thereof. For example, in some embodiments, theradius (r) value for an RCF of the side wall may be based upon the meanof the maximum and minimum possible radii or all possible radii alongthe length of the side wall of the chamber. In some embodiments, theradius for an exemplary centrifugal chamber sold by Biosafe AG for usewith the Sepax® system (e.g., A-200/F) is at or about 2.6 cm or at orabout 2.7 cm. In such an exemplary chamber, the radius for determiningRCF at the interface between the cell layer(s) and the liquid duringrotation in such a chamber may be calculated by adding the exact orapproximate radial distance between the internal side wall of the cavityand the chamber occupied by cells of the layer(s) during rotation. Suchvalue may be calculated or approximated using known methods, forexample, based on the diameter of one of the cells being processedand/or the average diameter among such cells, for example, duringrotation of the chamber. Such value may be based on the full size of thecell but typically will take into account impact on the relative volumeoccupied by each cell of the rotation or force itself, which generallyspeaking will reduce such volume. In some examples, the approximatedvalue is determined using the size of a nucleus of the cell (or averagethereof).

Thus, RCF or average RCF during a particular spin in a particularchamber or device may be calculated for a given point or area based onthe revolutions per minute (rpm) and the distance between that point andthe axis of rotation using well-known methods. Revolutions per minute(rpm) may be determined for various devices and chambers using knownmethods, for example, using a tachometer appropriate for the particulardevice, system, or chamber. For example, in some embodiments a hand-heldphoto or laser tachometer may be used, e.g., in combination withreflective tape, in the case of a centrifuge, system, or device with awindow from the environment to the chamber or cavity, such as theSepax®, which is clear or otherwise permits the passage of light betweenthe tachometer to the chamber. For opaque systems, other tachometers maybe used such as vibrating reed type tachometers.

As is understood by those in the art, when used in the context ofvarious vessels and containers, such as chambers, plates, tubes andbags, used in cell processing and centrifugation and materials thereof,rigid generally describes an object, portion thereof, or material whichsubstantially holds its shape and/or volume when placed in anenvironment, such as under a degree of force, temperature, or othercondition, in which one would ordinarily expect to be present in thecourse of using the object. For example, it is understood in the artthat rigid centrifugal chambers and tubes such as those made of hardplastic are distinguishable from flexible vessels such as cellprocessing and cell culture bags, such as bags made of soft plastics andrubbers, e.g., fluoro ethylene propylene and similar materials, theshape of which changes when pressure is applied manually or by pullingin liquid or gas, causing the bag to expand. Thus, in some embodiments,rigid materials include hard plastic, metal, carbon fiber, composites,ceramics, and glass, and/or are distinguished from flexible materialssuch as soft rubber, silicone, and plastics used in making flexiblebags, the shape and volume of which is easily changed by ordinarypressure, e.g., manual pressure or the filling of a vessel with liquidunder ambient temperature or ordinary conditions.

For example, in some embodiments, the rigid centrifugal chamber and/orportion(s) or material(s) thereof, such as the rigid side wall orportion thereof that surrounds the central cavity, is able to hold itsshape and/or volume and/or does not rupture or break in a way that itwould no longer contain liquid or gas, under particular conditions. Insome embodiments, such conditions include manual pressure, such aspressure capable of being applied by human hand. In some embodiments,such conditions include specified centrifugal forces, such as at a force(RCF), e.g., effective force, at the internal surface of the side wallof the cavity, of greater than at or about 200 g, greater than at orabout 300 g, or greater than at or about 500 g, such as greater than ator about 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g; or at leastat or about 1000 g, 1500 g, 2000 g, or 2500 g, 3000 g, or 3200 g, suchas at or about 2100 or 2200 g. In some embodiments, the environmentincludes particular conditions, such as temperatures down to at or about−80° C. and/or up to physiological temperatures or temperatures at whichcells remain viable, and/or higher, such as temperatures of 18° C. to42° C., such as 22° C. to 39° C., for example at least 25° C.±2° C. or37° C.±2° C.

As is understood in the art, describing an object as rigid orsubstantially rigid does not exclude the possibility that any change inshape or volume of an object or material would ever occur, such as underexcessive or unexpected force. For example, under excessive force orextreme environmental conditions, such as those well outside thoseordinarily used in connection with the transduction methods describedherein.

The chamber generally includes at least one opening, such as an inlet,an outlet, and/or an inlet/outlet, to permit substances to pass betweenthe cavity or other portion of the chamber and other spaces. Forexample, such opening(s) generally are included in at least one of thewalls of the chamber. The chamber generally includes at least one inletand at least one outlet, which in some embodiments may be the sameopening (inlet/outlet), through which liquid and/or gas may be takeninto and expressed from the cavity. The opening is generally associatedwith another environment via a channel, e.g., tubing line or system oftubing lines, in some embodiments, such as via one or more connectors.

In some embodiments, the chamber is included as part of and/or integralto a system, such as a closed or partially closed system, which furtherincludes additional components, such as tubing lines, connectors, andcontainers. In some embodiments, the chamber is pre-connected to one ormore of the additional components, directly and/or indirectly. Such achamber may be provided as part of a pre-assembled kit, e.g., a kitpackaged for single, sterile, use in connection with the providedmethods. In some embodiments, various components are packagedseparately, for example, to allow for custom configurations in which auser connects and arranges the components for a particular embodiment ofthe processing methods.

The components typically include at least one tubing line, and generallya set or system of tubing lines, and at least one connector. Exemplaryconnectors include valves, ports, spikes, welds, seals, and hose clamps.The connectors and/or other components may be aseptic, for example, topermit the entire process to be carried out in a closed, sterile system,which can eliminate or reduce the need for clean rooms, sterilecabinets, and/or laminar flow systems.

In some embodiments, the at least one tubing line includes a series oftubing lines. Tubing can be made of a plastic, such as polycarbonate,and may be of various sizes and/or volumes, generally designed to permitflow of the desired liquid/gas at the appropriate rate, and connectionwith the chamber and/or other components. The series of tubing linesgenerally allows for the flow of liquids and gases between the chamberand/or one or more components of the system, such as the othercontainers, facilitated in some aspects by connectors. In someembodiments, the system includes tubing lines connecting each of thevarious components to at least one other of the components, where liquidis permitted to flow between each of the containers, such as bags, andthe chamber, which may be permitted or stopped by the configuration ofvarious connectors, such as valves, and/or clamps.

In some aspects, the connectors are such that they may be placed in ordirected to alternative configurations, respectively blocking, allowing,and/or directing the flow of fluids and gases through variouscomponents, such as between various containers and through certaintubing lines connecting various components, such as rotational and gatevalves. In other embodiments, certain connectors and/or other componentshave a single configuration which permits, directs, or blocks passage ofliquid or gas, such as seals, caps, and/or open ports or channels.Various components in the system may include valves, ports, seals, andclamps. Valves can include rotational valves, such as stopcocks, rotaryvalves, and gate valves. Valves can be arranged in a manifold array oras a single multiport rotational valve. Ports may include Luer ports orspike ports. Seals may include O-rings, gaskets, adhesive seals, andcouplings. Clamps may include pinch clamps.

Other components of a system include containers capable of holding orstoring liquids and/or gases. The containers can include bags, vials,boxes, syringes, bulbs, tanks, bottles, beakers, buckets, flasks, andtubing lines. Such components can hold compositions used in and producedby the methods, including byproducts and interim products and waste.Such compositions may include liquid, including buffers, growth media,transduction media, water, diluents, washes, and/or saline, and may alsoinclude the cells, virus, and/or other agents for use in the processingsteps, such as transduction. The containers also may include wastecontainers, and containers holding one or more output product, such as aproduct containing cells selected and/or transduced by one or moreprocessing steps of the methods herein.

In some embodiments of the systems, a plurality of containers can besterilely connected at one or more positions on the tubing line of thesystem. The containers can be connected simultaneously and/orsequentially during methods of cell processing in the providedembodiments. In some embodiments, the containers are detachable orremovable from the connectors, such that the containers can be removedfrom the system and/or replaced by another container at the sameposition for use with the system. In some embodiments, not all connectorpositions of a system are connected to a container, such that the systemcan contain empty connectors. In some such embodiments, a closed systemis maintained by operation of one or more stopcocks, valves or clamps,either manually or automatically, to close communication between atubing line and an empty connector, e.g. port. In some embodiments, aclosed system is maintained by sealing or detaching an empty connector,e.g. port.

In some embodiments of the systems, such as the exemplary systemsdepicted in FIG. 5, FIG. 7 or FIG. 11, containers can be operablyconnected to tubing lines, such as through a connector, at positionscorresponding to an Input Bag position, Diluent Bag 1 position, aDiluent Bag 2 position, a Waste Bag position, and/or an Output Bagposition. With reference to the Figures, the designation of thesepositions is for exemplification only, and is not meant to limit theparticular type of container or content of the container that can beconnected at a position. Also, in embodiments of the provided methods,not all positions of the system, such as depicted in the Figures, needto be utilized in performing the processing steps of the providedmethods. In some such embodiments, a tubing line servicing an emptyconnector, e.g. port, can be disengaged or closed by operation of astopcock or valve. In some embodiments, an empty connector can be sealedor detached.

In some embodiments, the system, such as a closed system, is sterile. Insome embodiments, all connections of components of the system, such asbetween tubing line and a container via a connector, are made understerile conditions. In some embodiments, connections are made underlaminar flow. In some embodiments, connections are made using a sterileconnection device that produces sterile connections, such as sterilewelds, between a tubing and a container. In some embodiments, a sterileconnection device effects connection under thermal condition high enoughto maintain sterility, such as temperatures of at least 200° C., such asat least 260° C. or 300° C.

In some embodiments, the system may be disposable, such as a single-usekit. In some embodiments, a single-use kit can be utilized in aplurality of cycles of a process or processes, such as at least 2, 3, 4,5 or more times, for example, in processes that occur in a continuous ora semi-continuous manner. In some embodiments, the system, such as asingle-use kit, is employed for processing of cells from a singlepatient.

Exemplary centrifugal chambers include those produced and sold byBiosafe SA, including those for use with the Sepax® and Sepax® 2 system,including an A-200/F and A-200 centrifugal chambers and various kits foruse with such systems. Exemplary chambers, systems, and processinginstrumentation and cabinets are described, for example, in U.S. Pat.Nos. 6,123,655, 6,733,433 and Published U.S. patent application,Publication No.: US 2008/0171951, and published international patentapplication, publication no. WO 00/38762, the contents of each of whichare incorporated herein by reference in their entirety. Depending on theparticular process (e.g. dilution, wash, transduction, formulation), itis within the level of a skilled artisan to choose a particular kit thatis appropriate for the process. Exemplary kits for use with such systemsinclude, but are not limited to, single-use kits sold by BioSafe SAunder product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2.

In some embodiments, the system comprises a series of containers, e.g.,bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber.In some embodiments, the containers, such as bags, include one or morecontainers, such as bags, containing the cells to be transduced and theviral vector particles, in the same container or separate containers,such as the same bag or separate bags. In some embodiments, the systemfurther includes one or more containers, such as bags, containingmedium, such as diluent and/or wash solution, which is pulled into thechamber and/or other components to dilute, resuspend, and/or washcomponents and/or compositions during the methods. The containers can beconnected at one or more positions in the system, such as at a positioncorresponding to an input line, diluent line, wash line, waste lineand/or output line.

Exemplary systems for use in embodiments of the provided methods forcarrying out one or more or all part of the process are depicted in FIG.5, FIG. 7 and FIG. 11. In one exemplary embodiment as shown in FIG. 5,the centrifugal chamber (1) is at least generally cylindrical and isrotatable around an axis of rotation. The chamber includes an end wall(13) and a rigid side wall (14), and the movable member, which is apiston (2). The internal surfaces of the end wall (13), rigid side wall(14), and piston (2) collectively define the boundaries of the internalcavity (7) of the chamber. The cavity (7) is of variable volume and iscoaxial with the chamber, and is designed to contain the liquid and/orgas that is included within the chamber during the processing steps. Thepiston (2) is axially movable within the chamber (1) to vary the volumeof the internal cavity (7). The chamber further includes an inlet/outletopening (6) to permit flow of liquid and gas in and out of the cavity inat least some configurations of the system. The opening (6) is operablylinked with a series of tubing lines (3) and connectors, includingstopcock valves (4), which are capable of controlling movement of fluidand/or gas between the various components of the system. The series oftubing lines (3) further are linked with various additional containers,which in the depicted configuration include bags labeled as Input Bag,Diluent Bags 1 and 2, a Waste Bag, and an Output Bag. Clamps (5) may beopened and closed to permit and block movement of fluid through theindicated portions of the series of tubing lines (3), permitting flowbetween various components of the system. In some embodiments, eachcontainer is operably connected to a tubing line via a port, such as aluer port or spike port. As an example, with reference to FIG. 5, ateach point that a container is shown, in some aspects, the container isconnected indirectly via a port.

While FIG. 5 shows connection of a container at each position or line,in an alternative embodiment, in some aspects, a container is notconnected at each position or line of the system. In some embodiments ofprovided systems, a port is available at each position or line forconnection, and a container is connected to all positions or lines orless than all positions or lines. In some embodiments, not all connectorpositions of a system are connected to a container, such that the systemcan contain empty connectors at each position or line.

In some embodiments, the system, such as the system shown in FIG. 5, caninclude a sterile or microbial filter. Exemplary of such a system isshown in FIG. 7, which depicts a filter (15). In some embodiments, thefilter includes a filtration membrane having a pore size that blockspassage of microbial organisms, such as bacteria or viruses. In someembodiments, the pore size is between 0.1 μm to 0.45 μm, such as between0.1 μm to 0.22 μm, such as about or 0.20 μm. In some embodiments, themembrane is composed of nitrocellulose (cellulose nitrate), celluloseacetate, regenerated cellulose, polyamide, polytetrafluorethylene (PTFE)or polyethersulfone (PES). In some embodiments, the filter includes acap (16) to close or seal the membrane of the filter from exposure tothe environment outside of the closed system. In some embodiments, thecap is closed or non-vented. In some embodiments, the cap is detachable.In some embodiments, the cap is fitted to the filter by a luer lockfitting. As described in more detail below, in some embodiments, thefilter can be used to effect passage of gas, such as air, to and fromthe chamber of the system. In some such embodiments, the passage of airis maintained under sterile or microbial-free conditions.

In one embodiment, the Input Bag includes cells for processing by theprovided methods, such as transduction. In one embodiment, Diluent Bag 1includes viral vector particles containing the vector with which totransduce the cells. Thus, in some embodiments, the input compositioncontaining the viral vector particles and cells is generated byeffecting intake of fluid from the Input Bag and effecting intake offluid from Diluent Bag 1. In some embodiments, Diluent Bag 2 containswash solution. The Output Bag generally is designed to take in the cellsfollowing one or more of the processing steps, such as by transfer ofthe output composition from the cavity of the chamber to the Output Bagafter incubation with viral vector particles. Thus, in some embodiments,the Output Bag contains transferred cells transduced with and/or inwhich transduction is initiated with the viral vector particles. In someembodiments, the processing comprises transduction of the cells with aviral vector.

In some embodiments, a multi-way manifold (17) can be used to operablyconnect one or a plurality of containers to the system via a pluralityof ports (18) connected to a manifold of tubing lines. The multi-waymanifold can contain a series of tubing lines that feed to theinlet/outlet of the chamber to permit flow between the chamber and theconnected container or containers. In some such embodiments, themanifold connects a plurality of containers, such as at least 2, 3, 4,5, 6, 7, 8 or more containers, at the same position or line on thesystem. In some embodiments, all ports of the multi-way manifold (17)are connected to a container, such as a bag. In some embodiments, lessthan all of the ports of the multi-way manifold (17) are connected to acontainer, such as a bag, such that a container is connected at lessthan the total number of port positions, for example less than 8, 7, 6,5, 4, 3, or 2 containers, such as bags, are connected. In someembodiments, the tubing lines associated with the manifold can contain aclamp or stopcock, which can be opened or closed to control movementthrough the line into the container as necessary. The multi-way manifold(17) can be connected to any of the positions or lines available on thesystem, such to a position or line designated an input line, diluentline, wash line, waste line and/or output line.

In some embodiments, exemplary of a multi-way manifold (17) for linkinga container or containers is shown in FIG. 11 for connecting one or aplurality of containers, such as one or a plurality of bags, for exampleone or a plurality of output Bags. As shown, in some embodiments, amulti-way manifold (17) can be connected to an output position or line,which includes a series of manifold tubing lines that each end with aconnector, such as a port (18), for operable connection to a container,such as a bag. One or more of the ports, such as all of the ports orless than all of the ports, can be connected to a container. In oneembodiment as exemplified in FIG. 11, up to 3 containers can beconnected to each tubing line of the manifold via a port. In otherembodiments, up to 1, 2, 3, 4, 5, 6, 7, or 8 containers, such as Outputbags, can be connected at the output line. In some embodiments, one or aplurality of clamps (5) associated with tubing lines, such as themanifold tubing line, may be opened or closed to permit or control themovement of liquid into one or more of the plurality of Output Bags. Insome embodiments, a single clamp can control movement of liquid into allOutput Bags simultaneously. In some embodiments, the movement of liquidinto each of the plurality of bags is separately regulated by a clampoperably connected to a tubing line associated with only one respectivecontainer, such that the movement of liquid into the respectivecontainer can be made separable from the movement of liquid into allother containers. In some embodiments, movement of liquid into eachcontainer, such as each bag, for example each Output bag, can be made tobe sequential.

In some embodiments, the system is included with and/or placed intoassociation with other instrumentation, including instrumentation tooperate, automate, control and/or monitor aspects of the variousprocessing steps performed in the system. This instrumentation in someembodiments is contained within a cabinet.

In some embodiments, the instrumentation includes a cabinet, whichincludes a housing containing control circuitry, a centrifuge, a cover,motors, pumps, sensors, displays, and a user interface. An exemplarydevice is described in U.S. Pat. Nos. 6,123,655, 6,733,433 and US2008/0171951.

The control circuitry in some aspects monitors and communicatesinformation and instructions to and from the other instrumentation andvarious components of the system. In some embodiments, the cabinetcontains a user interface device, comprising a display and an inputdevice, such as a keyboard, a mouse, or a touchscreen. The userinterface displays information from the control circuitry, allows theuser to stop and start a process or steps, such as to effect atransduction protocol. The interface may also prompt the user to inputsettings for variables used by the control circuitry during a processstep, such as a transduction protocol. Such variables may include volumeof various solutions to be added and/or removed from the variouscontainers and/or the cavity of the chamber, time/duration ofsedimentation, centrifugation, agitation, mixing, and/or other processsteps, rotational force, piston movement, and/or program selection.

The instrumentation generally further includes a centrifuge, into whichthe centrifuge chamber is placed in order to effect rotation of thechamber. In some embodiments, the centrifuge chamber is engaged with arotary drive unit on the centrifuge apparatus, such that the chamber isrotatable about an axis of rotation. In some embodiments, a cover closeson top of the centrifuge chamber and holds the chamber in place. In someembodiments, the cover includes two semi-circular disks that can rotateon a hinge. An exemplary centrifuge and cover are described in U.S. Pat.No. 6,123,655 or 6,733,433. The centrifuge locks the centrifuge chamberinto place and rotates the centrifuge chamber by contacting thechamber's sides or ends.

In some embodiments, a sensor or an array of sensors in the centrifugecan measure the rotational speed of the centrifuge chamber, the positionof the movable member, or the volume contained within the internalcavity. Sensors outside of the centrifuge can detect the color and flowrate of liquid and gas flowing to and from the centrifuge chamber.Sensors can also detect an empty tubing or centrifuge chamber. Sensorsinclude optical sensors, such as those described in U.S. Pat. Nos.6,123,655, 6,733,433 and US 2008/0171951. In some embodiments, theinformation from the sensor or sensors can be received by controlcircuitry. Based on the information transmitted, the control circuitry,in some embodiments, can effect changes to one or more of the rotationalspeed of the centrifuge chamber, the position of the movable member, thevolume contained in the cavity, the orientation of one or more valves,ports, seals or clamps, and other processes of the centrifuge, chamberor system.

In some embodiments, the cabinet includes a motor or array of motors.The motors can communicate information with the control circuitry, whichcan operate or adjust the motors.

In some embodiments, the motor or array of motors can rotate thecentrifuge chamber within the centrifuge. The control circuitry canstart, stop, or adjust the speed of the motors rotating the centrifugechamber within the centrifuge.

In some embodiments, the motors or array of motors can move the movablemember within the centrifuge chamber. Moving the movable member variesthe volume of the internal cavity, causing the intake or expression ofliquid or gas to or from the internal cavity.

In some embodiments, the motors or array of motors can operate thevalves, ports, seals, and clamps described herein. The control circuitrycan cause the motors to open, close, or direct fluid to or from acontainer or the centrifuge chamber through the series of tubing.

In some embodiments, the motor or motors is an electrical motor,pneumatic motor or hydraulic motor. In some embodiments, the cabinetincludes an electrical motor for operating some aspects and a pneumaticmotor for operating other aspects. In some embodiments, the cabinetincludes an electrical motor for centrifugation and a pneumatic motorfor controlling movement of the movable member.

III. Transfer of Viral Nucleic Acids to Cells, e.g., by Transduction

In some embodiments, the processing step(s) of the methods include thosefor transfer of viral particles to the cells, such as viral vectorsencoding recombinant products to be expressed in the cells. The viralvector particles generally include a genome containing recombinantnucleic acids such as transgenes encoding such products. In someembodiments, the viral vector particles encode a recombinant receptor,such as a chimeric antigen receptor (CAR), whereby transduction of cellscan generate recombinant receptor (e.g. CAR)-expressing cells. Transferof the nucleic acid from the viral vector to the cells may use any of anumber of known methods. Transfer is typically by transduction.Alternative methods for transferring viral vectors to cells includetransposons and/or electroporation. Such processing steps can beperformed in a centrifugal chamber according to embodiments of theprovided methods. In some embodiments, the centrifugal chamber isintegral to a closed system, such that such processing steps areperformed in a closed system.

The transfer is generally carried out by transduction. The methods forviral transfer, e.g., transduction, generally involve at leastinitiation of transduction by incubating in a centrifugal chamber aninput composition comprising the cells to be transduced and viral vectorparticles containing the vector, under conditions whereby cells aretransduced or transduction is initiated in at least some of the cells inthe input composition, wherein the method produces an output compositioncomprising the transduced cells.

In some embodiments, the cells for transduction and/or transduced cellscontain immune cells, such as T cells, for use in adoptiveimmunotherapy. In some embodiments, prior to the incubation of cellswith viral vector particles, the cells for transduction are obtained bymethods that include isolating, such as selecting, a particular subsetof cells present in a biological sample. Methods related to isolationand selection of cells for transduction, and the resulting cells, aredescribed below. In some embodiments, prior to initiation of theprocesses for transduction, T cells are activated, such as bycultivation and stimulation as described below. In some embodiments, oneor more of all or a part of the steps related to isolation, e.g.selection, and activation also can be carried out in the cavity of acentrifugal chamber according to provided embodiments as describedbelow.

In some embodiments, the viral vector particles used in aspects of thetransduction method are any suitable for transduction of the cells, suchas an immune cell, for example a T cell. In some embodiments, the viralvector particles are retroviral vector particles, such as lentiviralvector particles or gammaretroviral vector particles. In some suchembodiments, the viral vector particle contains a genome comprising arecombinant nucleic acid, i.e. a recombinant viral vector. Exemplary ofsuch viral vector particles are described below.

The input composition (the composition that contains the viral vectorparticles and cells during the transduction step) may further includeone or more additional agents, such as those to promote transductionefficiency, such as polycations including protamine (e.g. protaminesulfate), hexadimethrine bromide (POLYBRENE®, Abbott Laboratories Corp),and CH-296 (RETRONECTIN®, Clontech). In some embodiments, the polycationcan be present in the input composition at a final concentration of 1μg/mL to 100 μg/mL, such as 5 μg/mL to 50 μg/mL. The composition mayalso include media, including cell culture medium including mediumdesigned for culture of the cell type to be processed, such ashematopoietic stem cell medium, e.g., serum free medium.

In the provided methods, all or a part of the processing steps fortransduction of cells can occur in the centrifugal chamber, such asunder centrifugation or rotation. In some such embodiments, the inputcomposition containing the cells and the viral vector particles areprovided to or taken into the internal cavity of the centrifugalchamber. In some embodiments, the input composition is incubated underconditions comprising rotation of the centrifugal chamber. In someembodiments, the rotation can be effected at relative centrifugal forcesgreater than can be achieved using flexible plastic bags or plasticmulti-well plates.

Greater transduction efficiency is achieved in some embodiments in partdue to the ability of the methods to carry out the transduction at agreater relative centrifugal force (RCF) compared with other methods forprocessing cells on large scales. For example, certain available methodsfor processing cells on a large scale, e.g., greater than 50 or 100 mLvolume, using flexible bags, may only permit centrifugation at arelative centrifugal force of no more than 200, 500, or 1000 g. Byallowing centrifugation at greater acceleration or relative force, e.g.,at or about or at least at or about 1000, 1500, 2000, 2100, 2200, 2500,3000 g, 3200 g or 3600 g, the provided methods can improve or permitco-sedimentation of virus and cells in the composition duringtransduction, improving the rate of virus-to-cell interactions, therebyimproving transduction.

The methods generally are capable of conducting the transduction on alarge scale. Thus, the input composition incubated during thetransduction and/or output composition may contain at least a certainvolume and/or number of cells. In some embodiments, the liquid volume ofthe input composition, or the liquid volume during at least a pointduring the incubation, is at least or greater than about 10 mL, 20 mL,30 mL, 40 mL, 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400mL, 450 mL, or 500 mL. In some embodiments, the input composition, thetransduced composition, and/or the total cells transduced by the methodsinclude at least at or about 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, or1×10¹⁰ cells. In some embodiments, for at least a portion of theincubation, the vessel in which the cells are transduced, e.g., thecentrifugal chamber or cavity thereof, contains at least at or about1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, or 1×10¹⁰ cells. Such numbers andvolume may also apply to other processing steps carried out in thesystem, e.g., in the cavity of the chamber, such as cell separationand/or washing steps.

In some embodiments, in describing the various processes steps in acavity of the centrifugal chamber, including processes for transduction,such as preparation of the input composition, or other process asdescribed in subsequent sections, reference to any volume is a targetvolume. In some embodiments, the exact volumes utilized in various steps(e.g. wash, dilution or formulation) can vary from a desired targetvolume, due to, in some aspects, dead volumes in a tubing line, primingof lines, sensitivity of a sensor, user control, and other factorsassociated with maintaining or monitoring a volume. The methods canpermit precise control of volumes, such as by, in some aspects,inclusion of a sensor as part of the circuitry associated with thesystem. In some embodiments, volumes vary by no more than 10% of adesired target volume, such as no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%,2% or 1%. In some embodiments, volumes are within 2 mL or 3 mL of atarget volume and/or vary by no more than 2 mL or 3 mL of a targetvolume.

In some embodiments, the processing steps are carried out by combiningthe cells and the viral vector particles to generate an inputcomposition. In aspects of the method, the composition of cells andviral vector particles are prepared in a manner so that the resultingcombined input composition has a low ratio of total liquid volume tointernal surface area of the cavity of the centrifugal chamber. In someembodiments, the total liquid volume is sufficient to cover or justexceed a volume of cells present as a monolayer on the internal surfaceof the cavity after rotation of the centrifugal chamber, whileminimizing the liquid thickness covering the cells. In some embodiments,reducing the liquid thickness can reduce the sedimentation time requiredfor contacting of the viral vector particles with the cells because theviral vector particles have less of a distance to travel and/or aresubjected to less resistance from the viscous medium.

In some embodiments, advantages such as improved transduction efficiencyare due at least in part to the ability to use a relatively lower volumeof liquid per volume of cells, cell number, or cell pellet size, duringprocesses of transduction, such as during rotation, particularlycompared with other methods for large-scale production.

In some embodiments, the liquid volume of the input composition(containing cells and viral vector particles) present in the vessel,e.g., cavity, during rotation is no more than about 0.5, 1, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 milliliters (mL) per square inch ofthe internal surface area of the cavity during the rotation or themaximum internal surface area of the cavity.

In particular embodiments, the average liquid volume of the inputcomposition present in the vessel, e.g., cavity, in which transductionis initiated, such as the average of the liquid volume of all processesperformed in a cycle of the method, is no more than about 0.5, 1, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 milliliters (mL) per squareinch of the internal surface area of the cavity during the incubation orof the maximum internal surface area of the cavity. In some embodiments,the maximum liquid volume of the input composition (containing cells andviral vector particles) present in the vessel, e.g., cavity, in whichtransduction is initiated, such as the maximum of the liquid volume ofall processes performed in a cycle of the method, is no more than about0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 milliliters (mL)per square inch of the internal surface area of the cavity of thecentrifugal chamber. In some embodiments, the liquid volume, such as theliquid volume of the input composition, present in the vessel, e.g.,cavity, during rotation is no more than 50%, such as no more than 40%,no more than 30%, no more than 20% or no more than 10% of the volume ofthe internal surface area of the cavity during rotation or the maximuminternal surface area of the cavity. In some embodiments, the remainderof the volume can be gas, such as air.

In some embodiments, the total liquid volume of the input composition(containing cells and viral vector particles) in the centrifugal chamberduring incubation, such as during rotation, is at least 5 mL or at least10 mL but is no more than 220 mL, such as no more than 200 mL. In someembodiments, the liquid volume of the input composition duringincubation, such as during rotation, is no more than 100 mL, 90 mL, 80mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20 mL. In aspects of theprovided method, the input composition is prepared at such a totalvolume to achieve a desired concentration, amount and/or ratio of cellsand viral vector particles, such as described below.

In some embodiments, the methods permit the user to control the ratio ofcells to surface of the cavity, e.g., by varying the volume of thecavity and/or number of cells added. In some embodiments, this allowsreduction of the layer of cells (e.g., cell pellet) on the surface ofthe cavity compared to other methods, particularly those available forlarge-scale transduction under centrifugal force, such as those carriedout in centrifuge bags. In some embodiments, the ability to control thethickness of the layer of cells in the cavity of the centrifugal chamberduring the transduction can lead to increased transduction efficiencyunder otherwise comparable conditions and/or a lack of increased copynumber with increased virus transduction efficiency.

In some embodiments, the cells in the provided methods are present inthe cavity in at or about a single monolayer, or no more than at orabout 1.5 or 2-fold more than a single monolayer, or not substantiallythicker than a monolayer, during the incubation for transduction undercentrifugal force. This reduction during centrifugation can facilitateand improve interactions between the virus and cells and avoid increasesin viral copy number (VCN) which can occur particularly in the contextof high relative virus or infectious units (IU), for example, when outeror upper layers of cells are preferentially transduced.

In some embodiments, the input composition contains at least 1 millioncells per cm² of the internal surface area of the cavity during at leasta portion of said incubation, such as during rotation of the inputcomposition in the centrifugal chamber. In some embodiments, the inputcomposition contains at least 2 million cells per cm², 3 million cellsper cm², 4 million cells per cm², 5 million cells per cm², 6 millioncells per cm², 7 million cells per cm², 8 million cells per cm², 9million cells per cm², 10 million cells per cm² or 20 million cells percm² of the internal surface area of the cavity during at least a portionof said incubation, such as during rotation of the input composition inthe centrifugal chamber. In some embodiments, the internal surface areaof the cavity during at least a portion of said incubation, such asduring rotation, is at least at or about 1×10⁹ μm² or is at least at orabout 1×10¹⁰ μm².

In some embodiments, the total number of cells in the input compositionduring at least a portion of said incubation, such as during rotation ofthe input composition in the centrifugal chamber, is at least 10×10⁶cells, 20×10⁶ cells, 30×10⁶ cells, 40×10⁶ cells, 50×10⁶ cells, 60×10⁶cells, 70×10⁶ cells, 80×10⁶ cells, 100×10⁶ cells, 200×10⁶ cells, 300×10⁶cells or 400×10⁶ cells.

In some embodiments, processing steps in the closed cavity of acentrifugal system also can be used to process the cells, such asactivated cells, prior to transduction. In some embodiments, theprocessing can include dilution or concentration of the cells to adesired concentration or number. In some embodiments, the processingsteps can include a volume-reduction to thereby increase theconcentration of cells as desired. In some embodiments, the processingincludes exchange of a medium into a medium acceptable or desired fortransduction.

In some embodiments, the input composition comprises a certain ratio ofcopies of the viral vector particles or infectious units (IU) thereof,per total number of cells (IU/cell) in the input composition or totalnumber of cells to be transduced. For example, in some embodiments, theinput composition includes at or about or at least at or about 1, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, or 60 IU of the viral vector particles perone of the cells.

In certain embodiments, the ability to use a higher IU in the presentmethods provides advantages compared to other methods. Under otherwiseidentical conditions, use of a higher IU/cell ratio generally leads to ahigher transduction efficiency, or does so up to a certain upper levelof IU/cell at which the corresponding increase in efficiency mayplateau. Nonetheless, with certain available methods, increasing theIU/cell and thus the transduction efficiency also leads to an increasein vector copy number (VCN), which can present safety risks and may notmeet regulatory standards.

In some embodiments, with the provided methods, average VCN amongtransduced cells in the output composition, such as cells containing theviral vector or cells expressing a molecule encoded by the viral vector,does not increase with an increase in IU/cell in the input composition.In some embodiments, in the provided methods, the average VCN amongtransduced cells decreases with an increased IU/cell ratio in the inputcomposition.

In some embodiments, the titer of viral vector particles is between orbetween about 1×10⁶ IU/mL and 1×10⁸ IU/mL, such as between or betweenabout 5×10⁶ IU/mL and 5×10⁷ IU/mL, such as at least 6×10⁶ IU/mL, 7×10⁶IU/mL, 8×10⁶ IU/mL, 9×10⁶ IU/mL, 1×10⁷ IU/mL, 2×10⁷ IU/mL, 3×10⁷ IU/mL,4×10⁷ IU/mL, or 5×10⁷ IU/mL.

In some embodiments, the input composition contains a concentration ofviral vector particles during at least a portion of said incubation,such as during rotation of the input composition in the centrifugalchamber, that has a certain ratio of copies of the viral vectorparticles or infectious units (IU) thereof, per total number of cells(IU/cell) in the input composition or total number of cells to betransduced per total liquid volume of the input composition presentduring at least a portion of said incubation, such as during rotation,i.e. IU/cell/mL. In some embodiments, the input composition includes atleast 0.01 IU, 0.05 IU, 0.1 IU, 0.5 IU or 0.1 IU of the viral vectorparticles per one of the cells per mL of the liquid volume of the inputcomposition during at least a portion of said incubation, such as duringrotation.

In some embodiments, the step of creating the input composition (cellsand viral vector particles) can be performed in the centrifugal chamber.In some embodiments, the step of creating the input composition isperformed outside the centrifugal chamber. Thus, the term “inputcomposition” is not meant to imply that the entire composition is takeninto the respective vessel, e.g., tube, bag, or cavity, at once, or toexclude the pulling in of parts of the composition from differentvessels or lines. Input compositions may include those formed by pullingin two different compositions into the chamber's cavity and mixing thetwo, thereby creating the input composition.

The input composition may be taken into or otherwise transferred to thevessel in which the incubation, such as rotation, takes place from thesame container or from more than one separate containers. For example,the input composition may be taken into the chamber by pulling in acomposition containing the cells and another composition containing theviral vector particles, which may be done sequentially orsimultaneously. Alternatively, the input composition containing theviral vector particles and cells is taken into the cavity or othervessel in which the transduction is to be carried out.

In some embodiments, where the transduction is carried out in theinternal cavity of the centrifugal chamber, this is achieved by allowingonly a certain portion of the cavity to include the liquid inputcomposition. This may be achieved, for example, by pulling in air or gasinto a portion of the cavity, and/or by including one or more solidobject in a space within the cavity, such as an internal space. In someembodiments, this can minimize or reduce the total liquid volume of saidinput composition present in said cavity during incubation, such asduring rotation, of said centrifugal chamber per square inch of theinternal surface area of the cavity compared to the absence of gas inthe cavity and/or absence of one or more solid objects in the space ofthe cavity. In this way, compared with other methods, in which diffusionof virus through a large volume of liquid compared to volume of cellsmay limit efficacy of transduction, the provided methods can beadvantageous. Thus, whereas in some embodiments, the input compositionoccupies all or substantially all of the volume of the internal cavityduring at least a portion of the incubation, in some embodiments, duringat least a portion of the incubation, the input composition occupiesonly a portion of the volume of the internal cavity during saidincubation.

In some such embodiments, the volume of the cavity during this at leasta portion of the incubation may further include a gas taken into saidcavity by the one or more opening, e.g., inlet, in the cavity, such asprior to or during said incubation. In some embodiments of the method,the air is sterilized or is sterile air. In some embodiments, the air isfree of or substantially free of microbial contaminants or otherpotentially pathogenic agents.

In some embodiments, providing or taking in gas, such as air, can beeffected in any manner that permits passage of air into the internalcavity of the centrifugal chamber, such as, in some aspects, withoutcompromising the sterility of the closed system. In some embodiments,gas, such as air, can be added to a container under sterile conditions,and the container can be sterilely connected at a position on the systemfor transfer into the chamber. In some embodiments, the addition of gas,such as air, to the container, such as a bag, is effected under laminarflow conditions, such as in a biological safety cabinet or hood. In somesuch embodiments, the gas, such as air, is added to the containertogether with a liquid volume, such as a liquid volume containing acomposition of cells and/or a liquid volume containing a composition ofviral vector particles. Hence, in some embodiments, providing or takingin gas, such as air, into the internal cavity of the chamber occurstogether or simultaneously with the providing or intake of one or bothof the cells or viral vector particles that make up the inputcomposition.

In some embodiments, the providing or taking in gas, such as air, intothe chamber, is achieved using a syringe that can be attached to anyluer lock associated with the system, and, that is operably connected tothe internal cavity of the centrifugal chamber. In some embodiments, airis transferred into the syringe under sterile conditions, such as underlaminar flow. In some embodiments, the syringe is a sterile syringe,such as, in some aspects, a syringe containing a movable plunger that isnot exposed to the surrounding non-sterile environment. In someembodiments, the syringe contains a filter at its end to effect steriletransfer of gas, such as air, into the internal cavity of the chamber.

In some embodiments, providing or taking in gas, such as air, into theinternal cavity of the chamber is achieved by the use of a filteroperably connected to the internal cavity of the chamber via a steriletubing line. In some such embodiments, the filter is a sterile ormicrobial filter as described with reference to an exemplary system,such as in some aspects, a filter as exemplified in FIG. 7. In someembodiments, a device is connected to the filter, such as via a luerlock connection, to transfer the air. In some such embodiments, thedevice is a syringe, pump, or other infusion device. In someembodiments, the gas is air, and the intake of air through the filter isdirectly from the surrounding environment. In some embodiments, thefilter contains a cap, such as a non-vented cap, that is removable ordetachable to control transfer of air into filter as desired.

Hence, in some embodiments, the methods include providing or taking in aliquid input composition and a volume of gas, such as air, into theinternal cavity of the chamber. The volume of gas, such as air, that isprovided or taken in is a function of the volume of compositioncontaining cells and composition containing viral vector particles thatmake up the input composition. In some embodiments, the volume of gas isthe difference between the total volume of the internal cavity and theliquid volume of the input composition. In some embodiments, the totalvolume of gas and liquid is no more than 200 mL, such that the volume ofgas provided or taken in to the internal cavity is the differencebetween 200 mL and the liquid volume of the input composition (cells andviral vector particles).

In an exemplary aspect of the provided methods, the method oftransduction includes providing to an internal cavity of a closedcentrifugal chamber system, in which the internal cavity has a surfacearea of at least at or about 1×10⁹ μm² or at least at or about 1×10¹⁰μm², a composition containing at least or about 50×10⁶ cells in a volumethat is no more than 100 mL. In some embodiments, the cell compositioncontains at least or about 100×10⁶ cells or at least or about 200×10⁶cells in a volume that is no more no more than 50 mL, 40 mL, 30 mL, 20mL, 10 mL or 5 mL. In some embodiments, prior to providing the cells tothe internal cavity, the composition of cells are diluted orconcentrated to a volume of no more than 100 mL, such as no more no morethan 50 mL, 40 mL, 30 mL, 20 mL, 10 mL or 5 mL. In addition to the cellcomposition, the method also includes providing, in some aspects, acomposition containing viral vector particles in an amount that is atleast 1 IU/cell in a volume so that the total liquid volume, includingfrom the composition containing cells, is less than the maximum volumeof the internal cavity of the centrifugal chamber, such as no more than200 mL, thereby generating the input composition. In some embodiments,the composition containing viral vector particles is provided in anamount that is at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4IU/cell, 2.8 IU/cell, 3.2 IU/cell or 3.6 IU/cell. In some embodiments,the total liquid volume of the input composition is less than 100 mL,less than 90 mL, less than 80 mL, less than 60 mL, less than 40 mL, lessthan 20 mL. Optionally, the method also can include providing gas, suchas air up to the total volume of the internal cavity, for example, sothat the total volume occupied in the internal cavity of the centrifugalchamber is up to or about 200 mL.

In some embodiments, the composition containing cells and compositioncontaining viral vector particles, and optionally air, can be combinedor mixed prior to providing the compositions to the cavity. In someembodiments, the composition containing cells and composition containingviral vector particles, and optionally air, are provided separately andcombined and mixed in the cavity. In some embodiments, a compositioncontaining cells, a composition containing viral vector particles, andoptionally air, can be provided to the internal cavity in any order. Inany of such some embodiments, a composition containing cells and viralvector particles is the input composition once combined or mixedtogether, whether such is combined or mixed inside or outside thecentrifugal chamber and/or whether cells and viral vector particles areprovided to the centrifugal chamber together or separately, such assimultaneously or sequentially.

In some embodiments, intake of the volume of gas, such as air, occursprior to the incubation, such as rotation, in the transduction method.In some embodiments, intake of the volume of gas, such as air, occursduring the incubation, such as rotation, in the transduction method.

In some embodiments, the liquid volume of the cells or viral vectorparticles that make up the input composition, and optionally the volumeof air, can be a predetermined volume. The volume can be a volume thatis programmed into and/or controlled by circuitry associated with thesystem.

In some embodiments, intake of the input composition, and optionallygas, such as air, is controlled manually, semi-automatically and/orautomatically until a desired or predetermined volume has been takeninto the internal cavity of the chamber. In some embodiments, a sensorassociated with the system can detect liquid and/or gas flowing to andfrom the centrifuge chamber, such as via its color, flow rate and/ordensity, and can communicate with associated circuitry to stop orcontinue the intake as necessary until intake of such desired orpredetermined volume has been achieved. In some aspects, a sensor thatis programmed or able only to detect liquid in the system, but not gas(e.g. air), can be made able to permit passage of gas, such as air, intothe system without stopping intake. In some such embodiments, anon-clear piece of tubing can be placed in the line near the sensorwhile intake of gas, such as air, is desired. In some embodiments,intake of gas, such as air, can be controlled manually.

In aspects of the provided methods, the internal cavity of thecentrifuge chamber is subjected to high speed rotation. In someembodiments, rotation is effected prior to, simultaneously, subsequentlyor intermittently with intake of the liquid input composition, andoptionally air. In some embodiments, rotation is effected subsequent tointake of the liquid input composition, and optionally air. In someembodiments, rotation is by centrifugation of the centrifugal chamber ata relative centrifugal force at the inner surface of side wall of theinternal cavity and/or at a surface layer of the cells of at or about orat least at or about 800 g, 1000 g, 1100 g, 1500, 1600 g, 1800 g, 2000g, 2200 g, 2500 g, 3000 g, 3500 g or 4000 g. In some embodiments,rotation is by centrifugation at a force that is greater than or about1100 g, such as by greater than or about 1200 g, greater than or about1400 g, greater than or about 1600 g, greater than or about 1800 g,greater than or about 2000 g, greater than or about 2400 g, greater thanor about 2800 g, greater than or about 3000 g or greater than or about3200 g.

In some embodiments, the method of transduction includes rotation orcentrifugation of the input composition, and optionally air, in thecentrifugal chamber for greater than or about 5 minutes, such as greaterthan or about 10 minutes, greater than or about 15 minutes, greater thanor about 20 minutes, greater than or about 30 minutes, greater than orabout 45 minutes, greater than or about 60 minutes, greater than orabout 90 minutes or greater than or about 120 minutes. In someembodiments, the input composition, and optionally air, is rotated orcentrifuged in the centrifugal chamber for greater than 5 minutes, butfor no more than 60 minutes, no more than 45 minutes, no more than 30minutes or no more than 15 minutes.

In some embodiments, the method of transduction includes rotation orcentrifugation of the input composition, and optionally air, in thecentrifugal chamber for between or between about 10 minutes and 60minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30minutes and 60 minutes or 45 minutes and 60 minutes, each inclusive, andat a force at the internal surface of the side wall of the internalcavity and/or at a surface layer of the cells of at least or greaterthan or about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g,2000 g, 2200 g, 2400 g, 2800 g, 3200 g or 3600 g.

In some embodiments, the method includes effecting expression from theinternal cavity of the centrifugal chamber an output composition, whichis the resulting composition of cells incubated with viral vectorparticles under conditions that include rotation or centrifugation inthe centrifugal chamber in any of the above embodiments as described. Inaspects of the method, the output composition includes cells transducedwith, or in which transduction has been initiated with, a viral vector.In some embodiments, the expression of the output composition is to anoutput bag that is operably linked as part of a closed system with thecentrifugal chamber. In some embodiments, expression of the outputcomposition is subsequent to the rotation or centrifugation. In someembodiments, expression of the output composition is simultaneous withor partly simultaneous with the rotation or centrifugation, such as in asemi-continuous or continuous process.

In some embodiments, the gas, such as air, in the cavity of the chamberis expelled from the chamber. In some embodiments, the gas, such as air,is expelled to a container that is operably linked as part of the closedsystem with the centrifugal chamber. In some embodiments, the containeris a free or empty container. In some embodiments, the air, such as gas,in the cavity of the chamber is expelled through a filter that isoperably connected to the internal cavity of the chamber via a steriletubing line. In some embodiments, the air is expelled using manual,semi-automatic or automatic processes. In some embodiments, air isexpelled from the chamber prior to, simultaneously, intermittently orsubsequently with expressing the output composition containing incubatedcells and viral vector particles, such as cells in which transductionhas been initiated or cells have been transduced with a viral vector,from the cavity of the chamber.

In some embodiments, the transduction and/or other incubation isperformed as or as part of a continuous or semi-continuous process. Insome embodiments, a continuous process involves the continuous intake ofthe cells and viral vector particles, e.g., the input composition(either as a single pre-existing composition or by continuously pullinginto the same vessel, e.g., cavity, and thereby mixing, its parts),and/or the continuous expression or expulsion of liquid, and optionallyexpelling of gas (e.g. air), from the vessel, during at least a portionof the incubation, e.g., while centrifuging. In some embodiments, thecontinuous intake and continuous expression are carried out at least inpart simultaneously. In some embodiments, the continuous intake occursduring part of the incubation, e.g., during part of the centrifugation,and the continuous expression occurs during a separate part of theincubation. The two may alternate. Thus, the continuous intake andexpression, while carrying out the incubation, can allow for a greateroverall volume of sample to be processed, e.g., transduced.

In some embodiments, the incubation is part of a continuous process, themethod including, during at least a portion of the incubation, effectingcontinuous intake of said input composition into the cavity duringrotation of the chamber and during a portion of the incubation,effecting continuous expression of liquid and, optionally expelling ofgas (e.g. air), from the cavity through the at least one opening duringrotation of the chamber.

In some embodiments, the semi-continuous incubation is carried out byalternating between effecting intake of the composition into the cavity,incubation, expression of liquid from the cavity and, optionallyexpelling of gas (e.g. air) from the cavity, such as to an outputcontainer, and then intake of a subsequent (e.g., second, third, etc.)composition containing more cells and other reagents for processing,e.g., viral vector particles, and repeating the process. For example, insome embodiments, the incubation is part of a semi-continuous process,the method including, prior to the incubation, effecting intake of theinput composition into the cavity through said at least one opening, andsubsequent to the incubation, effecting expression of fluid from thecavity; effecting intake of another input composition comprising cellsand the viral vector particles into said internal cavity; and incubatingthe another input composition in said internal cavity under conditionswhereby said cells in said another input composition are transduced withsaid vector. The process may be continued in an iterative fashion for anumber of additional rounds. In this respect, the semi-continuous orcontinuous methods may permit production of even greater volume and/ornumber of cells.

In some embodiments, a portion of the transduction incubation isperformed in the centrifugal chamber, which is performed underconditions that include rotation or centrifugation.

In some embodiments, the method includes an incubation in which afurther portion of the incubation of the cells and viral vectorparticles is carried out without rotation or centrifugation, whichgenerally is carried out subsequent to the at least portion of theincubation that includes rotation or centrifugation of the chamber. Insome such embodiments, the further incubation is effected underconditions to result in integration of the viral vector into a hostgenome of one or more of the cells. It is within the level of a skilledartisan to assess or determine if the incubation has resulted inintegration of viral vector particles into a host genome, and hence toempirically determine the conditions for a further incubation. In someembodiments, integration of a viral vector into a host genome can beassessed by measuring the level of expression of a recombinant protein,such as a heterologous protein, encoded by a nucleic acid contained inthe genome of the viral vector particle following incubation. A numberof well-known methods for assessing expression level of recombinantmolecules may be used, such as detection by affinity-based methods,e.g., immunoaffinity-based methods, e.g., in the context of cell surfaceproteins, such as by flow cytometry. In some examples, the expression ismeasured by detection of a transduction marker and/or reporterconstruct. In some embodiments, nucleic acid encoding a truncatedsurface protein is included within the vector and used as a marker ofexpression and/or enhancement thereof.

In some embodiments, the further incubation is carried out in thecentrifuge chamber, but without rotation. In some embodiments, thefurther incubation is carried out outside of the centrifuge chamber. Insome embodiments, the further incubation is effected at temperaturesgreater than room temperature, such as greater than or greater thanabout 25° C., such as generally greater than or greater than about 32°C., 35° C. or 37° C. In some embodiments, the further incubation iseffected at a temperature of at or about 37° C.±2° C., such as at atemperature of at or about 37° C. In some embodiments, the furtherincubation is for a time between or about between 1 hour and 48 hours, 4hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours,inclusive.

In some embodiments, the further incubation occurs in a closed system.In some embodiments, after expression of the output composition from thechamber, such as into a container (e.g. bag), the container containingthe output composition is incubated for a further portion of time. Insome embodiments, the container, such as bag, is incubated at atemperature of at or about 37° C.±2° C. for a time between or aboutbetween 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hoursor 12 hours and 24 hours, inclusive.

In some embodiments, the methods effect transduction of a certain numberor percentage of the cells in the input and/or output (transduced)composition, or subset thereof. For example, in some embodiments, atleast 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 75% of the total cells (or of a particular target cell type, suchas T cells) in the input composition and/or in the output (e.g.,transduced) composition, are transduced with said viral vector and/orexpress the recombinant gene product encoded thereby. In someembodiments, the methods of transduction result in an output compositionin which at least 2.5%, at least 5%, at least 6%, at least 8%, at least10%, at least 20%, at least 25%, at least 30%, at least 40%, at least50%, or at least 75% of the total cells, such as T cells, in thecomposition are transduced with the viral vector and/or express therecombinant gene product encoded thereby.

In some embodiments, the methods are capable of achieving such at leasta particular transduction efficiency under certain conditions. Forexample, in some embodiments, where the input composition includes thevirus and cells at a ratio of from or from about 1 infectious unit (IU)per one of the cells to 10 IU per one of the cells, such as at or about1 infectious units (IU) per one of the cells, or at or about 2 IU perone of the cells, at or about 5 IU per one of the cells, or at or about10 IU per one of the cells, the method is capable of producing atransduced composition in which at least 10%, at least 25%, at least30%, at least 40%, at least 50%, or at least 75% of the cells in saidtransduced composition generated by the method comprise, e.g., have beentransduced with, the recombinant viral vector. Transduction of the cellsmay be detected by detecting the presence of recombinant nucleic acid,e.g., transgene, included in the vector or product thereof in the cell.In some embodiments, the product is detected on the surface of the cell,indicating the cell has been successfully transduced. In someembodiments, detection of transduction involves detection of atransduction marker, such as another transgene or product included forthe purposes of marking transduced cells, and/or other selection marker.

In some embodiments, the output composition resulting from thetransduction methods includes a particular average or mean number ofcopies of the transduced vector per cell (vector copy number (VCN)). VCNmay be expressed in terms of the number of copies in a single cell.Alternatively, it may be expressed as an average number over a totalcell population or composition, such as the output or transducedcomposition (including any non-transduced cells within the composition,which would not include any copies of the vector). Alternatively, VCNmay be expressed in terms of average copy number only among thetransduced cells. In some embodiments, among all the cells in thetransduced or output composition produced by the methods, the averageVCN is no more than at or about 10, 5, 4, 2.5, 1.5, or 1. In someembodiments, among the cells in the transduced or output compositionthat contain the recombinant viral vector or express the recombinantgene product, the average VCN is no more than at or about 4, 3, 2, 2.5,1.5, or 1.

Also provided are compositions produced by any of the above methods. Insome embodiments, the compositions contain at least 1×10⁷ cells or 5×10⁷cells, such as at least 1×10⁸ cells, 2×10⁸ cells, 4×10⁸ cells, 6×10⁸,8×10⁸ cells or 1×10⁹ cells, in which at least a plurality of cells aretransduced with the recombinant viral vector. In some embodiments, thecells are T cells.

In some embodiments, by practice of the methods provided herein, it ispossible to produce an output composition containing a plurality oftransduced cells in high number, such as, in some aspects, a number thatcan achieve a therapeutically effective dosage of T cells for use inadoptive immunotherapy. In some embodiments, this can be achieved notonly because of the ability to transduce cells on a large scale, butalso, in some aspects, by repeating the process in a continuous orsemi-continuous manner.

In contrast, existing methods in the art in which transduction isperformed on a smaller scale, such as in plates, requires large scaleexpansion of the cells after transduction in order to achieve numbers ofcells necessary to obtain a therapeutically effective dosage. Expansionof cells, such as T cells, with one or more stimulating agents canactivate the cells and/or alter the phenotype of the cells, such as byresulting in the generation of effector cells with an exhausted T cellphenotype. For example, activation or stimulation of T cells can resultin a change in differentiation or activation state of T cells that mayresult and/or lead to reduced persistence in vivo when geneticallyengineered cells are administered to a subject. Among changes indifferentiation state that may occur include, in some cases, loss of anaïve phenotype, loss of memory T cell phenotypes, and/or the generationof effector cells with an exhausted T cell phenotype. Exhaustion of Tcells may lead to a progressive loss of T cell functions and/or indepletion of the cells (Yi et al. (2010) Immunology, 129:474-481). Tcell exhaustion and/or the lack of T cell persistence is a barrier tothe efficacy and therapeutic outcomes of adoptive cell therapy; clinicaltrials have revealed a correlation between greater and/or longer degreeof exposure to the antigen receptor (e.g. CAR)-expressing cells andtreatment outcomes.

In some embodiments, in the methods provided herein it is not necessaryto stimulate and/or activate cells subsequent to transduction to thesame extent as is necessary in other known methods in the art. In someembodiments, subsequent to transduction, the cells in the compositionare not subject to expansion in the presence of a stimulating agent(e.g. a cytokine, such as IL-2) and/or are not incubated at atemperature greater than or about 30° C. or greater than or about 37° C.for more than 24 hours. In some embodiments, at least 30%, 40%, 50%,60%, 70%, 80%, or 90% of the T cells in the composition and/ortransduced T cells in the output composition comprise high surfaceexpression of CD69 or TGF-beta-II. In some embodiments, at least 30%,40%, 50%, 60%, 70%, 80%, or 90% of the T cells or transduced T cells inthe composition comprise no surface expression of CD62L and/or comprisehigh expression of CD25, ICAM, GM-CSF, IL-8 and/or IL-2.

In some embodiments, engineered cells, such as cells transduced with theviral vectors encoding recombinant products to be expressed in thecells, of the output composition produced by the above method, or by amethod that includes a further processing step, such as to generate aformulated composition, exhibit increased persistence when administeredin vivo to a subject. In some embodiments, the persistence of a providedcells, such as receptor, e.g., CAR, -expressing cells, in the subjectupon administration is greater as compared to that which would beachieved by alternative methods of transduction, such as those involvingadministration of cells genetically engineered by methods involvingsmaller scale transduction in which T cells are activated and/orstimulated to expand prior to and/or subsequent to transduction toachieve a number of cells that is a therapeutically effective dose. Forexample, in some aspects, the persistence of provided cells, such ascells produced by the provided methods, is greater as compared to thatwhich would be achieved by administration of a population of geneticallyengineered recombinant receptor (e.g. CAR)-expressing in which at least30%, 40%, 50%, 60%, 70%, 80%, or 90% have a lower level of expression ofCD69 or TGF-beta II. In some embodiments, the persistence of providedcells, such as cells produced by the provided methods, is greatercompared to that which would be achieved by administration of apopulation of genetically engineered recombinant receptor (e.g.CAR)-expressing in which at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%exhibit surface expression of CD62L and/or comprise low surfaceexpression of CD25, ICAM, GM-CSF, IL-8 and/or IL-2.

In some embodiments, the persistence is increased at least or about atleast 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold,90-fold, 100-fold or more.

In some embodiments, the degree or extent of persistence of administeredcells can be detected or quantified after administration to a subject.For example, in some aspects, quantitative PCR (qPCR) is used to assessthe quantity of cells expressing the recombinant receptor (e.g.,CAR-expressing cells) in the blood or serum or organ or tissue (e.g.,disease site) of the subject. In some aspects, persistence is quantifiedas copies of DNA or plasmid encoding the receptor, e.g., CAR, permicrogram of DNA, or as the number of receptor-expressing, e.g.,CAR-expressing, cells per microliter of the sample, e.g., of blood orserum, or per total number of peripheral blood mononuclear cells (PBMCs)or white blood cells or T cells per microliter of the sample. In someembodiments, flow cytometric assays detecting cells expressing thereceptor generally using antibodies specific for the receptors also canbe performed. Cell-based assays may also be used to detect the number orpercentage of functional cells, such as cells capable of binding toand/or neutralizing and/or inducing responses, e.g., cytotoxicresponses, against cells of the disease or condition or expressing theantigen recognized by the receptor. In any of such embodiments, theextent or level of expression of another marker associated with therecombinant receptor (e.g. CAR-expressing cells) can be used todistinguish the administered cells from endogenous cells in a subject.

In some embodiments, by minimizing T cell activation and/or stimulation,the provided embodiments can result in genetically engineered T cellsthat are more potent for use in adoptive immunotherapy methods, due, insome aspects, to increased persistence. In some embodiments, theincreased potency and/or increased persistence of the provided cells,such as cells produced by any of the provided methods, permits methodsof administering cells at lower dosages. Such methods can minimizetoxicity that can occur from adoptive immunotherapy methods.

Other Cell Processing Events

In some embodiments, in addition to and/or alternatively to thetransduction steps, the processing methods of the provided methodsinclude other processing steps and methods, such as for the isolation,separation, selection, cultivation (e.g., stimulation of the cells, forexample, to induce their proliferation and/or activation), washing,suspension, dilution, concentration, and/or formulation of the cells. Insome embodiments, at least a portion of one or more other processingsteps and/or at least a portion of a plurality of the steps are carriedout in whole or in part within the cavity of a centrifugal chamber, suchas the same or different centrifugal chamber as used in the methods oftransduction. In some embodiments, all or a portion of such one or moreother processing steps are carried out in the closed system containing acentrifugal chamber, such as in a sterile closed system.

In some embodiments, the methods include one or more of (a) washing abiological sample containing cells (e.g., a whole blood sample, a buffycoat sample, a peripheral blood mononuclear cells (PBMC) sample, anunfractionated T cell sample, a lymphocyte sample, a white blood cellsample, an apheresis product, or a leukapheresis product) in a cavity ofa chamber, (b) isolating, e.g. selecting, from the sample a desiredsubset or population of cells (e.g., CD4+ or CD8+ T cells) in a cavityof a chamber, for example, by incubation of cells with a selection orimmunoaffinity reagent for immunoaffinity-based separation; c)incubating the isolated, such as selected cells, with viral vectorparticles, such as in accord with methods described above and d)formulating the transduced cells, such as in a pharmaceuticallyacceptable buffer, cryopreservative or other suitable medium. In someembodiments, the methods can further include (e) stimulating cells in acavity of a chamber by exposing cells to stimulating conditions, therebyinducing cells to proliferate. In some embodiments, the step ofstimulating the cells is performed prior to, during and/or subsequent tothe incubation of cells with viral vector particles. In someembodiments, one or more further step of washing or suspending step,such as for dilution, concentration and/or buffer exchange of cells, canalso be carried out prior to or subsequent to any of the above steps.

Thus, in some embodiments, the methods carry out one, more, or all stepsin the preparation of cells for clinical use, e.g., in adoptive celltherapy, without exposing the cells to non-sterile conditions andwithout the need to use a sterile room or cabinet. In some embodimentsof such a process, the cells are isolated, separated or selected,stimulated, transduced, washed, and formulated, all within a closedsystem. In some embodiments, the methods are carried out in an automatedfashion. In some embodiments, one or more of the steps is carried outapart from the centrifugal chamber system.

Samples

In some embodiments, the processing steps include isolation of cells orcompositions thereof from biological samples, such as those obtainedfrom or derived from a subject, such as one having a particular diseaseor condition or in need of a cell therapy or to which cell therapy willbe administered. In some aspects, the subject is a human, such as asubject who is a patient in need of a particular therapeuticintervention, such as the adoptive cell therapy for which cells arebeing isolated, processed, and/or engineered. Accordingly, the cells insome embodiments are primary cells, e.g., primary human cells. Thesamples include tissue, fluid, and other samples taken directly from thesubject, as well as samples resulting from one or more processing steps,such as separation, centrifugation, genetic engineering (e.g.transduction with viral vector), washing, and/or incubation. Thebiological sample can be a sample obtained directly from a biologicalsource or a sample that is processed. Biological samples include, butare not limited to, body fluids, such as blood, plasma, serum,cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organsamples, including processed samples derived therefrom.

In some aspects, the sample is blood or a blood-derived sample, or is oris derived from an apheresis or leukapheresis product. Exemplary samplesinclude whole blood, peripheral blood mononuclear cells (PBMCs),leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia,lymphoma, lymph node, gut associated lymphoid tissue, mucosa associatedlymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach,intestine, colon, kidney, pancreas, breast, bone, prostate, cervix,testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.Samples include, in the context of cell therapy, e.g., adoptive celltherapy, samples from autologous and allogeneic sources.

In some embodiments, isolation of the cells or populations includes oneor more preparation and/or non-affinity based cell separation steps. Insome examples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components. In someexamples, cells from the circulating blood of a subject are obtained,e.g., by apheresis or leukapheresis. The samples may containlymphocytes, including T cells, monocytes, granulocytes, B cells, othernucleated white blood cells, red blood cells, and/or platelets.

In some embodiments, the provided methods include processing, in wholeor in part, one or more of the samples in a closed system, such as in acentrifugal chamber. In some embodiments, the processing step caninvolve washing of the sample, e.g., blood cell-containing sample, fromthe subject, e.g., to remove the plasma fraction and/or replacing thecells in an appropriate buffer or media for subsequent processing stepsand/or performing a density-based cell separation methods, such as inthe preparation of white blood cells from peripheral blood by lysing thered blood cells and centrifugation through a Percoll or Ficoll gradient.Exemplary of such processing steps can be performed using a centrifugalchamber in conjunction with one or more systems associated with a cellprocessing system, such as a centrifugal chamber produced and sold byBiosafe SA, including those for use with the Sepax® or Sepax 2® cellprocessing systems.

Affinity-Based Selection

The processing steps (e.g., carried out in the centrifugal chamber) mayinclude isolation of cells from mixed populations and/or compositions,such as using one of various selection steps including density-based orother physical property-based separation methods and affinity-basedselection. In some embodiments, the methods include selection in whichall or a portion of the selection is carried out in the internal cavityof the centrifugal chamber, for example, under centrifugal rotation. Insome embodiments, incubation of cells with selection reagents, such asimmunoaffinity-based selection reagents, is performed in a centrifugalchamber. Such methods can offer certain advantages compared to otheravailable selection methods.

For example, immunoaffinity-based selection can depend upon a favorableenergetic interaction between the cells being separated and the moleculespecifically binding to the marker on the cell, e.g., the antibody orother binding partner on the solid surface, e.g., particle. In certainavailable methods for affinity-based separation using particles such asbeads, particles and cells are incubated in a container, such as a tubeor bag, while shaking or mixing, with a constant celldensity-to-particle (e.g., bead) ratio to aid in promoting energeticallyfavored interactions. Such approaches may not be ideal for use withlarge-scale production, for example, in that they may require use oflarge volumes in order to maintain an optimal or desiredcell-to-particle ratio while maintaining the desired number of cells.Accordingly, such approaches can require processing in batch mode orformat, which can require increased time, number of steps, and handling,increasing cost and risk of user error.

In some embodiments, by conducting such selection steps or portionsthereof (e.g., incubation with antibody-coated particles, e.g., magneticbeads) in the cavity of the centrifugal chamber, the user is able tocontrol certain parameters, such as volume of various solutions,addition of solution during processing and timing thereof, which canprovide advantages compared to other available methods. For example, theability to decrease the liquid volume in the cavity during theincubation can increase the concentration of the particles (e.g. beadreagent) used in the selection, and thus the chemical potential of thesolution, without affecting the total number of cells in the cavity.This in turn can enhance the pairwise interactions between the cellsbeing processed and the particles used for selection. In someembodiments, carrying out the incubation step in the chamber, e.g., whenassociated with the systems, circuitry, and control as described herein,permits the user to effect agitation of the solution at desired time(s)during the incubation, which also can improve the interaction.

In some embodiments, at least a portion of the selection step isperformed in a centrifugal chamber, which includes incubation of cellswith a selection reagent. In some aspects of such processes, a volume ofcells is mixed with an amount of a desired affinity-based selectionreagent that is far less than is normally employed when performingsimilar selections in a tube or container for selection of the samenumber of cells and/or volume of cells according to manufacturer'sinstructions. In some embodiments, an amount of selection reagent orreagents that is/are no more than 5%, no more than 10%, no more than15%, no more than 20%, no more than 25%, no more than 50%, no more than60%, no more than 70% or no more than 80% of the amount of the sameselection reagent(s) employed for selection of cells in a tube orcontainer-based incubation for the same number of cells and/or the samevolume of cells according to manufacturer's instructions is employed.

The incubation with a selection reagent or reagents, e.g., as part ofselection methods which may be performed in the chamber cavity, includeusing one or more selection reagents for selection of one or moredifferent cell types based on the expression or presence in or on thecell of one or more specific molecules, such as surface markers, e.g.,surface proteins, intracellular markers, or nucleic acid. In someembodiments, any known method using a selection reagent or reagents forseparation based on such markers may be used. In some embodiments, theselection reagent or reagents result in a separation that is affinity-or immunoaffinity-based separation. For example, the selection in someaspects includes incubation with a reagent or reagents for separation ofcells and cell populations based on the cells' expression or expressionlevel of one or more markers, typically cell surface markers, forexample, by incubation with an antibody or binding partner thatspecifically binds to such markers, followed generally by washing stepsand separation of cells having bound the antibody or binding partner,from those cells having not bound to the antibody or binding partner.

In some embodiments, for selection, e.g., immunoaffinity-based selectionof the cells, the cells are incubated in the cavity of the chamber in acomposition that also contains the selection buffer with a selectionreagent, such as a molecule that specifically binds to a surface markeron a cell that it desired to enrich and/or deplete, but not on othercells in the composition, such as an antibody, which optionally iscoupled to a scaffold such as a polymer or surface, e.g., bead, e.g.,magnetic bead, such as magnetic beads coupled to monoclonal antibodiesspecific for CD4 and CD8. In some embodiments, as described, theselection reagent is added to cells in the cavity of the chamber in anamount that is substantially less than (e.g. is no more than 5%, 10%,20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to theamount of the selection reagent that is typically used or would benecessary to achieve about the same or similar efficiency of selectionof the same number of cells or the same volume of cells when selectionis performed in a tube with shaking or rotation. In some embodiments,the incubation is performed with the addition of a selection buffer tothe cells and selection reagent to achieve a target volume withincubation of the reagent of, for example, 10 mL to 200 mL, such as atleast or about at least or about or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL,60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL. In someembodiments, the selection buffer and selection reagent are pre-mixedbefore addition to the cells. In some embodiments, the selection bufferand selection reagent are separately added to the cells. In someembodiments, the selection incubation is carried out with periodicgentle mixing condition, which can aid in promoting energeticallyfavored interactions and thereby permit the use of less overallselection reagent while achieving a high selection efficiency.

In some embodiments, the total duration of the incubation with theselection reagent is from or from about 5 minutes to 6 hours, such as 30minutes to 3 hours, for example, at least or about at least 30 minutes,60 minutes, 120 minutes or 180 minutes.

In some embodiments, the incubation generally is carried out undermixing conditions, such as in the presence of spinning, generally atrelatively low force or speed, such as speed lower than that used topellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g.at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm),such as at an RCF at the sample or wall of the chamber or othercontainer of from or from about 80 g to 100 g (e.g. at or about or atleast 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spinis carried out using repeated intervals of a spin at such low speedfollowed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.

In some embodiments, such process is carried out within the entirelyclosed system to which the chamber is integral. In some embodiments,this process (and in some aspects also one or more additional step, suchas a previous wash step washing a sample containing the cells, such asan apheresis sample) is carried out in an automated fashion, such thatthe cells, reagent, and other components are drawn into and pushed outof the chamber at appropriate times and centrifugation effected, so asto complete the wash and binding step in a single closed system using anautomated program.

In some embodiments, after the incubation and/or mixing of the cells andselection reagent and/or reagents, the incubated cells are subjected toa separation to select for cells based on the presence or absence of theparticular reagent or reagents. In some embodiments, the furtherselection is performed outside of the centrifugal chamber. In someembodiments, the separation is performed in the same closed system inwhich the centrifugal chamber is present and in which the incubation ofcells with the selection reagent was performed. In some embodiments,after incubation with the selection reagents, incubated cells, includingcells in which the selection reagent has bound, are expressed from thecentrifugal chamber, such as transferred out of the centrifugal chamber,into a system for immunoaffinity-based separation of the cells. In someembodiments, the system for immunoaffinity-based separation is orcontains a magnetic separation column. In some embodiments, prior toseparation, one or more other processing steps can be performed in thechamber, such as washing.

Such separation steps can be based on positive selection, in which thecells having bound the reagents, e.g. antibody or binding partner, areretained for further use, and/or negative selection, in which the cellshaving not bound to the reagent, e.g., antibody or binding partner, areretained. In some examples, both fractions are retained for further use.In some aspects, negative selection can be particularly useful where noantibody is available that specifically identifies a cell type in aheterogeneous population, such that separation is best carried out basedon markers expressed by cells other than the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types. In any of such examples, at least aportion of the further selection or selection steps is performed in acentrifugal chamber, which includes incubation of cells with a selectionreagent, as described above.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+,and/or CD45RO+ T cells, are isolated by positive or negative selectiontechniques. In some embodiments, such cells are selected by incubationwith one or more antibody or binding partner that specifically binds tosuch markers. In some embodiments, the antibody or binding partner canbe conjugated, such as directly or indirectly, to a solid support ormatrix to effect selection, such as a magnetic bead or paramagneticbead. For example, CD3+, CD28+ T cells can be positively selected usingCD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander, and/or ExpACT® beads).

In some embodiments, the process steps further include negative and/orpositive selection of the incubated and cells, such as using a system orapparatus that can perform an affinity-based selection. In someembodiments, isolation is carried out by enrichment for a particularcell population by positive selection, or depletion of a particular cellpopulation, by negative selection. In some embodiments, positive ornegative selection is accomplished by incubating cells with one or moreantibodies or other binding agent that specifically bind to one or moresurface markers expressed or expressed (marker+) at a relatively higherlevel (markerhigh) on the positively or negatively selected cells,respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+cytotoxic T cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8+ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (TCM) cells is carried outto increase efficacy, such as to improve long-term survival, expansion,and/or engraftment following administration, which in some aspects isparticularly robust in such sub-populations. See Terakura et al. (2012)Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In someembodiments, combining TCM-enriched CD8+ T cells and CD4+ T cellsfurther enhances efficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L−subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (TCM) cells isbased on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD 127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8+ population enriched forTCM cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (TCM) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8+ cell population or subpopulation, also is used to generate theCD4+ cell population or sub-population, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4+ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4+ T helper cells may be sorted into naïve, central memory, andeffector cells by identifying cell populations that have cell surfaceantigens. CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, orCD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− andCD45RO−.

In one example, to enrich for CD4+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinitymagnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).

In some aspects, the incubated sample or composition of cells to beseparated is incubated with a selection reagent containing small,magnetizable or magnetically responsive material, such as magneticallyresponsive particles or microparticles, such as paramagnetic beads(e.g., such as Dynalbeads or MACS® beads). The magnetically responsivematerial, e.g., particle, generally is directly or indirectly attachedto a binding partner, e.g., an antibody, that specifically binds to amolecule, e.g., surface marker, present on the cell, cells, orpopulation of cells that it is desired to separate, e.g., that it isdesired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. Many well-knownmagnetically responsive materials for use in magnetic separation methodsare known, e.g., those described in Molday, U.S. Pat. No. 4,452,773, andin European Patent Specification EP 452342 B, which are herebyincorporated by reference. Colloidal sized particles, such as thosedescribed in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat.No. 5,200,084 also may be used.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody—or other bindingpartner (e.g., streptavidin)-coated magnetic particles, are added. Incertain embodiments, streptavidin-coated magnetic particles are used inconjunction with biotinylated primary or secondary antibodies.

In some aspects, separation is achieved in a procedure in which thesample is placed in a magnetic field, and those cells havingmagnetically responsive or magnetizable particles attached thereto willbe attracted to the magnet and separated from the unlabeled cells. Forpositive selection, cells that are attracted to the magnet are retained;for negative selection, cells that are not attracted (unlabeled cells)are retained. In some aspects, a combination of positive and negativeselection is performed during the same selection step, where thepositive and negative fractions are retained and further processed orsubject to further separation steps.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS), e.g., CliniMACS systemsare capable of high-purity selection of cells having magnetizedparticles attached thereto. In certain embodiments, MACS operates in amode wherein the non-target and target species are sequentially elutedafter the application of the external magnetic field. That is, the cellsattached to magnetized particles are held in place while the unattachedspecies are eluted. Then, after this first elution step is completed,the species that were trapped in the magnetic field and were preventedfrom being eluted are freed in some manner such that they can be elutedand recovered. In certain embodiments, the non-target cells are labelledand depleted from the heterogeneous population of cells.

In some embodiments, the processing steps include expression from thecentrifugal chamber of cells incubated with one or more selectionreagents. In some embodiments, the cells can be expressed subsequent toand/or continuous with one or more washing steps, which can, in someaspects, be performed in the centrifugal chamber.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies,magnetizable particles or antibodies conjugated to cleavable linkers,etc. In some embodiments, the magnetizable particles are biodegradable.

Freezing and Cryopreservation

In some embodiments, the cells, such as selected cells, are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively.

In some embodiments, the cells, such as selected cells, can betransferred to cryopreservation media using a centrifugal chamber inconjunction with one or more systems associated with a cell processingsystem, such as a centrifugal chamber produced and sold by Biosafe SA,including those for use with the Sepax® or Sepax 2® cell processingsystems. In some embodiments, transfer to cryopreservation medium isassociated with one or more processing steps that can involve washing ofthe sample, e.g., selected cell sample, such as to remove the selectionmedia and/or replacing the cells in an appropriate cryopreservationbuffer or media for subsequent freezing.

In some embodiments, the cells are frozen, e.g., cryopreserved, eitherbefore, during, or after said methods for processing. In someembodiments, the freeze and subsequent thaw step removes granulocytesand, to some extent, monocytes in the cell population. The cells aregenerally then frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank.

Cultivation and Stimulation

In some embodiments, the processing steps (e.g., those carried out inthe chamber and/or closed system) include cultivation, stimulationand/or activation of cells, such as by incubation and/or culture ofcells. For example, in some embodiments, provided are methods forstimulating the isolated cells, such as selected cell populations. Insome embodiments, the processing steps include incubation of acomposition containing the cells, such as selected cells, where at leasta portion of the incubation is in a centrifugal chamber and/or othervessel, e.g., under stimulating conditions. The incubation may be priorto or in connection with genetic engineering, such as geneticengineering resulting from embodiments of the transduction methoddescribed above. In some embodiments, the stimulation results inactivation and/or proliferation of the cells, for example, prior totransduction.

In some embodiments, the processing steps include incubations of cells,such as selected cells, in which the incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation ofcells. In some embodiments, the compositions or cells are incubated inthe presence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

In some embodiments, the conditions for stimulation and/or activationcan include one or more of particular media, temperature, oxygencontent, carbon dioxide content, time, agents, e.g., nutrients, aminoacids, antibiotics, ions, and/or stimulatory factors, such as cytokines,chemokines, antigens, binding partners, fusion proteins, recombinantsoluble receptors, and any other agents designed to activate the cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell, such as agents suitable to deliver a primary signal, e.g., toinitiate activation of an ITAM-induced signal, such as those specificfor a TCR component, and/or an agent that promotes a costimulatorysignal, such as one specific for a T cell costimulatory receptor, e.g.,anti-CD3, anti-CD28, or anti-41-BB, for example, bound to solid supportsuch as a bead, and/or one or more cytokines. Among the stimulatingagents are anti-CD3/anti-CD28 beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander, and/or ExpACT® beads). Optionally, the expansion methodmay further comprise the step of adding anti-CD3 and/or anti CD28antibody to the culture medium. In some embodiments, the stimulatingagents include IL-2 and/or IL-15, for example, an IL-2 concentration ofat least about 10 units/mL. In some embodiments, incubation is carriedout in accordance with techniques such as those described in U.S. Pat.No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother.35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang etal. (2012) J Immunother. 35(9):689-701.

In some embodiments, the T cells are expanded by adding to thecomposition feeder cells, such as non-dividing peripheral bloodmononuclear cells (PBMC), (e.g., such that the resulting population ofcells contains at least about 5, 10, 20, or 40 or more PBMC feeder cellsfor each T lymphocyte in the initial population to be expanded); andincubating the culture (e.g. for a time sufficient to expand the numbersof T cells). In some aspects, the non-dividing feeder cells can comprisegamma-irradiated PBMC feeder cells. In some embodiments, the PBMC areirradiated with gamma rays in the range of about 3000 to 3600 rads toprevent cell division. In some aspects, the feeder cells are added toculture medium prior to the addition of the populations of T cells.

In some embodiments, the stimulating conditions generally include atemperature suitable for the growth of human T lymphocytes, for example,at least about 25 degrees Celsius, generally at least about 30 degrees,and generally at or about 37 degrees Celsius. Optionally, the incubationmay further comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+and/or CD8+ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

In some embodiments, at least a portion of the incubation with one ormore stimulating conditions or stimulatory agents, such as any describedabove, is performed in a centrifugal chamber. In some embodiments, atleast a portion of the incubation performed in a centrifugal chamberincludes mixing with a reagent or reagents to induce stimulation and/oractivation. In some embodiments, cells, such as selected cells, aremixed with a stimulating condition or stimulatory agent in thecentrifugal chamber. In some aspects of such processes, a volume ofcells is mixed with an amount of one or more stimulating conditions oragents that is far less than is normally employed when performingsimilar stimulations in a cell culture plate or other system.

In some embodiments, the stimulating agent is added to cells in thecavity of the chamber in an amount that is substantially less than (e.g.is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of theamount) as compared to the amount of the stimulating agent that istypically used or would be necessary to achieve about the same orsimilar efficiency of selection of the same number of cells or the samevolume of cells when selection is performed without mixing in acentrifugal chamber, e.g. in a tube or bag with periodic shaking orrotation. In some embodiments, the incubation is performed with theaddition of an incubation buffer to the cells and stimulating agent toachieve a target volume with incubation of the reagent of, for example,10 mL to 200 mL, such as at least or about at least or about or 10 mL,20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mLor 200 mL. In some embodiments, the incubation buffer and stimulatingagent are pre-mixed before addition to the cells. In some embodiments,the incubation buffer and stimulating agent are separately added to thecells. In some embodiments, the stimulating incubation is carried outwith periodic gentle mixing condition, which can aid in promotingenergetically favored interactions and thereby permit the use of lessoverall stimulating agent while achieving stimulating and activation ofcells.

In some embodiments, the total duration of the incubation with thestimulating agent is from or from about 1 hour and 72 hours, 1 hour and48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24hours, such as at least or about at least 6 hours, 12 hours, 18 hours,24 hours, 36 hours or 72 hours. In some cases, the total duration of theincubation in the centrifugal chamber is from or from about 5 minutes to6 hours, such as 30 minutes to 3 hours, for example, at least or aboutat least 30 minutes, 60 minutes, 120 minutes or 180 minutes. In somecases, a further portion of the incubation can be performed outside ofthe centrifugal chamber.

In some embodiments, the incubation generally is carried out undermixing conditions, such as in the presence of spinning, generally atrelatively low force or speed, such as speed lower than that used topellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g.at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm),such as at an RCF at the sample or wall of the chamber or othercontainer of from or from about 80 g to 100 g (e.g. at or about or atleast 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spinis carried out using repeated intervals of a spin at such low speedfollowed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.

In some embodiments, cells are incubated in a centrifugal chamber with acell stimulating agent or agents that is/are a cell-binding agent, suchas an antigen-binding reagent, such as antibody, that is able to induceintracellular signaling and/or cell proliferation. In some embodiments,cells are incubated with, including mixed with, anti-CD3/anti-CD28 beadsin a centrifugal chamber according to aspects of processes in theprovided methods.

In some embodiments, the processing steps include expression from thecentrifugal chamber of cells incubated, such as mixed with, one or morestimulatory conditions or stimulating agents. In some embodiments, oneor more other additional processing steps can be performed in thechamber, such as washing, which can be prior to, subsequent to and/orcontinuous with the stimulating incubation. In some embodiments, washingis performed prior to stimulation, such as on selected or thawed cells,to remove and replace media with a medium suitable for stimulation andcultivation of cells.

In some embodiments, expressed cells from the centrifugal chamber thathave been incubated, such as mixed with, one or more stimulatoryconditions or stimulating agents, are further incubated outside of thechamber. In some embodiments, the further incubation is effected attemperatures greater than room temperature, such as greater than orgreater than about 25° C., such as generally greater than or greaterthan about 32° C., 35° C. or 37° C. In some embodiments, the furtherincubation is effected at a temperature of at or about 37° C.±2° C.,such as at a temperature of at or about 37° C. In some embodiments, thefurther incubation is for a time between or about between 12 hours and96 hours, such as at least or at least about 12 hours, 24 hours, 36hours, 48 hours, 72 hours or 96 hours.

In some embodiments, the further incubation occurs in a closed system.In some embodiments, after expression from the chamber of the cellsincubated, such as mixed, with one or stimulatory conditions orstimulating agents, such as into a container (e.g. bag), the containercontaining the cells is incubated for a further portion of time. In someembodiments, the container, such as bag, is incubated at a temperatureof at or about 37° C.±2° C. for a time between or about between 1 hourand 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and24 hours, inclusive.

Formulation

In some embodiments, the process steps (e.g. carried out in thecentrifugal chamber and/or closed system) may include formulation ofcells, such as formulation of genetically engineered cells resultingfrom the provided transduction processing steps and/or one or more otherprocessing steps as described. In some embodiments, the provided methodsassociated with formulation of cells include processing transducedcells, such as cells transduced and/or expanded using the processingsteps described above, in a closed system, such as in or associated witha centrifugal chamber.

In some embodiments, the cells are formulated in a pharmaceuticallyacceptable buffer, which may, in some aspects, include apharmaceutically acceptable carrier or excipient. In some embodiments,the processing includes exchange of a medium into a medium orformulation buffer that is pharmaceutically acceptable or desired foradministration to a subject. In some embodiments, the processing stepscan involve washing the transduced and/or expanded cells to replace thecells in a pharmaceutically acceptable buffer that can include one ormore optional pharmaceutically acceptable carriers or excipients.Exemplary of such pharmaceutical forms, including pharmaceuticallyacceptable carriers or excipients, can be any described below inconjunction with forms acceptable for administering the cells andcompositions to a subject. The pharmaceutical composition in someembodiments contains the cells in amounts effective to treat or preventthe disease or condition, such as a therapeutically effective orprophylactically effective amount.

In some embodiments, the formulation buffer contains a cryopreservative.In some embodiments, the cell are formulated with a cyropreservativesolution that contains 1.0% to 30% DMSO solution, such as a 5% to 20%DMSO solution or a 5% to 10% DMSO solution. In some embodiments, thecryopreservation solution is or contains, for example, PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. In some embodiments, the cryopreservative solution is orcontains, for example, at least or about 7.5% DMSO. In some embodiments,the processing steps can involve washing the transduced and/or expandedcells to replace the cells in a cryopreservative solution.

In some embodiments, the processing can include dilution orconcentration of the cells to a desired concentration or number, such asunit dose form compositions including the number of cells foradministration in a given dose or fraction thereof. In some embodiments,the processing steps can include a volume-reduction to thereby increasethe concentration of cells as desired. In some embodiments, theprocessing steps can include a volume-addition to thereby decrease theconcentration of cells as desired.

In some embodiments, the processing includes adding a volume of aformulation buffer to transduced and/or expanded cells. In someembodiments, the volume of formulation buffer is from or from about 10mL to 1000 mL, such as at least or about at least or about or 50 mL, 100mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL or1000 mL.

Exemplary of such processing steps can be performed using a centrifugalchamber in conjunction with one or more systems or kits associated witha cell processing system, such as a centrifugal chamber produced andsold by Biosafe SA, including those for use with the Sepax® or Sepax 2®cell processing systems.

In some embodiments, the method includes effecting expression from theinternal cavity of the centrifugal chamber a formulated composition,which is the resulting composition of cells formulated in a formulationbuffer, such as pharmaceutically acceptable buffer, in any of the aboveembodiments as described. In some embodiments, the expression of theformulated composition is to a container, such as a bag that is operablylinked as part of a closed system with the centrifugal chamber. In someembodiments, the container, such as bag, is connected to a system at anoutput line or output position as exemplified in the exemplary systemsdepicted in FIG. 5 or FIG. 7.

In some embodiments, the closed system, such as associated with a cellprocessing system, such as centrifugal chamber, includes a multi-portoutput kit containing a multi-way tubing manifold associated at each endof a tubing line with a port to which one or a plurality of containerscan be connected for expression of the formulated composition. In someaspects, a desired number or plurality of output containers, e.g., bags,can be sterilely connected to one or more, generally two or more, suchas at least 3, 4, 5, 6, 7, 8 or more of the ports of the multi-portoutput. For example, in some embodiments, one or more containers, e.g.,bags can be attached to the ports, or to fewer than all of the ports.Thus, in some embodiments, the system can effect expression of theoutput composition into a plurality of output bags.

In some aspects, cells can be expressed to the one or more of theplurality of output bags in an amount for dosage administration, such asfor a single unit dosage administration or multiple dosageadministration. For example, in some embodiments, the output bags mayeach contain the number of cells for administration in a given dose orfraction thereof. Thus, each bag, in some aspects, may contain a singleunit dose for administration or may contain a fraction of a desired dosesuch that more than one of the plurality of output bags, such as two ofthe output bags, or 3 of the output bags, together constitute a dose foradministration.

Thus, the containers, e.g., bags, generally contain the cells to beadministered, e.g., one or more unit doses thereof. The unit dose may bean amount or number of the cells to be administered to the subject ortwice the number (or more) of the cells to be administered. It may bethe lowest dose or lowest possible dose of the cells that would beadministered to the subject.

In some embodiments, each of the containers, e.g., bags, individuallycomprises a unit dose of the cells. Thus in some embodiments, each ofthe containers comprises the same or approximately or substantially thesame number of cells. In some embodiments, the unit dose includes lessthan about 1×10⁸, less than about 5×10⁷, less than about 1×10⁶ or lessthan about 5×10⁵ of cells, per kg of the subject to be treated and/orfrom which the cells have been derived. In some embodiments, each unitdose contains at least or about at least 1×10⁶, 2×10⁶, 5×10⁶, 1×10⁷,5×10⁷, or 1×10⁸ engineered cells, total cells, T cells, or PBMCs. Insome embodiments, the volume of the formulated cell composition in eachbag is 10 mL to 100 mL, such as at least or about at least 20 mL, 30 mL,40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL or 100 mL.

In some embodiments, one or more of the plurality of output bags can beused for testing, such as for assessing transduction efficiency. Forexample, the transduction efficiency in some aspects may be assessed bymeasuring the level of expression of a recombinant protein, such as aheterologous protein, encoded by a nucleic acid contained in the genomeof the viral vector particle following transduction using embodiments ofthe provided methods. Thus, in some embodiments, the expression level ofrecombinant molecules may be assessed by any of a number of well-knownmethods such as detection by affinity-based methods, e.g.,immunoaffinity-based methods, e.g., in the context of cell surfaceproteins, such as by flow cytometry. In some aspects, the cellscontained in one or more of the plurality of containers, e.g., bags, istested for the expression level of recombinant molecules by detection ofa transduction marker and/or reporter construct. In other embodiments,expression is assessed using a nucleic acid encoding a truncated surfaceprotein included within the vector as a marker.

In some embodiments, all or substantially all of a plurality ofcontainers to which cells are expressed contain the same number of cellsand in the same or substantially the same concentration. In someembodiments, prior to expressing cells into one of a plurality ofcontainers, the tubing lines are primed.

IV. Cells and Compositions

Among the cells to be used in the methods, such as the processing steps,e.g., the transfer of viral nucleic acids, e.g., transduction, arecells, cell populations, and compositions.

The cells generally are mammalian cells, and typically are human cells.In some embodiments, the cells are derived from the blood, bone marrow,lymph, or lymphoid organs. In some aspects, the cells are cells of theimmune system, such as cells of innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets of Tcells or other cell types, such as whole T cell populations, CD4+ cells,CD8+ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. In some embodiments, themethods include isolating cells from the subject, preparing, processing,culturing, and/or engineering them, and re-introducing them into thesame subject, before or after cryopreservation, which, in some aspects,can be achieved in a closed system using one or more of the providedprocessing steps.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/orof CD8+ T cells are naïve T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(T_(SCM)), central memory T (T_(CM)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

V. Viral Vector Particles, Viral Vectors, and Encoded RecombinantProducts

The transduction methods generally involve transduction with viralvectors, such as those encoding recombinant products to be expressed inthe cells. The term “vector,” as used herein, refers to a nucleic acidmolecule capable of propagating another nucleic acid to which it islinked. The term includes the vector as a self-replicating nucleic acidstructure as well as the vector incorporated into the genome of a hostcell into which it has been introduced. Certain vectors are capable ofdirecting the expression of nucleic acids to which they are operativelylinked. Such vectors are referred to herein as “expression vectors.”Vectors include viral vectors, such as retroviral vectors, for examplelentiviral or gammaretroviral vectors, having a genome carrying anothernucleic acid and capable of inserting into a host genome for propagationthereof.

In some embodiments, a viral vector is transferred to a cell in avehicle that is a viral vector particle, which includes a virion thatencapsulates and/or packages a viral vector genome. In some suchembodiments, the genome of the viral vector typically includes sequencesin addition to the nucleic acid encoding the recombinant molecule thatallow the genome to be packaged into the virus particle.

In some embodiments, the viral vector contains a recombinant nucleicacid, such as a nucleic acid encoding a recombinant and/or heterologousmolecule, such as a recombinant or heterologous protein. In someembodiments, such as in aspects of the provided methods, transductionwith the viral vectors produces an output composition, cells of whichhave been transduced and express recombinant or genetically engineeredproducts of such nucleic acids. In some embodiments, the nucleic acidsare heterologous, i.e., normally not present in a cell or sampleobtained from the cell, such as one obtained from another organism orcell, which for example, is not ordinarily found in the cells beingtransduced and/or an organism from which such cell is derived. In someembodiments, the nucleic acids are not naturally occurring, such as anucleic acid not found in nature, including one comprising chimericcombinations of nucleic acids encoding various domains from multipledifferent cell types.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant virus or viral vector particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into cells, such as T cells, using recombinantlentiviral vectors or retroviral vectors, such as gamma-retroviralvectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi:10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46;Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al.,Trends Biotechnol. 2011 November 29(11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

The viral vectors generally include recombinant nucleic acids such astransgenes encoding recombinant products to be expressed by the cells.Recombinant products include recombinant receptors, including antigenreceptors such as functional non-TCR antigen receptors, e.g., chimericantigen receptors (CARs), and other antigen-binding receptors such astransgenic T cell receptors (TCRs). Also among the receptors are otherchimeric receptors.

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321,WO2013/071154, WO2013/123061 U.S. patent application publication numbersUS2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995,7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319,7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118,and European patent application number EP2537416, and/or those describedby Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila etal. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol.,2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, the antigen receptors include a CAR asdescribed in U.S. Pat. No. 7,446,190, and those described inInternational Patent Application Publication No.: WO/2014055668 A1.Examples of the CARs include CARs as disclosed in any of theaforementioned publications, such as WO2014031687, U.S. Pat. Nos.8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190,8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology,10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701;and Brentjens et al., Sci Transl Med. 2013 5(177). See alsoWO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S.Pat. Nos. 7,446,190, and 8,389,282. The chimeric receptors, such asCARs, generally include an extracellular antigen binding domain, such asa portion of an antibody molecule, generally a variable heavy (V_(H))chain region and/or variable light (V_(L)) chain region of the antibody,e.g., an scFv antibody fragment.

In some embodiments, the antibody portion of the recombinant receptor,e.g., CAR, further includes at least a portion of an immunoglobulinconstant region, such as a hinge region, e.g., an IgG4 hinge region,and/or a CH1/CL and/or Fc region. In some embodiments, the constantregion or portion is of a human IgG, such as IgG4 or IgG1. In someaspects, the portion of the constant region serves as a spacer regionbetween the antigen-recognition component, e.g., scFv, and transmembranedomain. The spacer can be of a length that provides for increasedresponsiveness of the cell following antigen binding, as compared to inthe absence of the spacer. Exemplary spacers, e.g., hinge regions,include those described in international patent application publicationnumber WO2014031687. In some examples, the spacer is or is about 12amino acids in length or is no more than 12 amino acids in length.Exemplary spacers include those having at least about 10 to 229 aminoacids, about 10 to 200 amino acids, about 10 to 175 amino acids, about10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids,about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20amino acids, or about 10 to 15 amino acids, and including any integerbetween the endpoints of any of the listed ranges. In some embodiments,a spacer region has about 12 amino acids or less, about 119 amino acidsor less, or about 229 amino acids or less. Exemplary spacers includeIgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4hinge linked to the CH3 domain.

This antigen recognition domain generally is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor. Thus, in some embodiments, the antigen-binding component(e.g., antibody) is linked to one or more transmembrane andintracellular signaling domains. In some embodiments, the transmembranedomain is fused to the extracellular domain. In one embodiment, atransmembrane domain that naturally is associated with one of thedomains in the receptor, e.g., CAR, is used. In some instances, thetransmembrane domain is selected or modified by amino acid substitutionto avoid binding of such domains to the transmembrane domains of thesame or different surface membrane proteins to minimize interactionswith other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD5, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively the transmembrane domain in some embodiments issynthetic. In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. In some embodiments,the linkage is by linkers, spacers, and/or transmembrane domain(s).

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In someembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

The receptor, e.g., the CAR, generally includes at least oneintracellular signaling component or components. In some embodiments,the receptor includes an intracellular component of a TCR complex, suchas a TCR CD3 chain that mediates T-cell activation and cytotoxicity,e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portionis linked to one or more cell signaling modules. In some embodiments,cell signaling modules include CD3 transmembrane domain, CD3intracellular signaling domains, and/or other CD transmembrane domains.In some embodiments, the receptor, e.g., CAR, further includes a portionof one or more additional molecules such as Fc receptor γ, CD8, CD4,CD25, or CD16. For example, in some aspects, the CAR or other chimericreceptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fcreceptor γ and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimericreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in some contexts, the CAR induces a function of a Tcell such as cytolytic activity or T-helper activity, such as secretionof cytokines or other factors. In some embodiments, a truncated portionof an intracellular signaling domain of an antigen receptor component orcostimulatory molecule is used in place of an intact immunostimulatorychain, for example, if it transduces the effector function signal. Insome embodiments, the intracellular signaling domain or domains includethe cytoplasmic sequences of the T cell receptor (TCR), and in someaspects also those of co-receptors that in the natural context act inconcert with such receptors to initiate signal transduction followingantigen receptor engagement.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In other embodiments, the CAR does not include a component forgenerating a costimulatory signal. In some aspects, an additional CAR isexpressed in the same cell and provides the component for generating thesecondary or costimulatory signal.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

In some aspects, the CAR includes a primary cytoplasmic signalingsequence that regulates primary activation of the TCR complex. Primarycytoplasmic signaling sequences that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signaling sequences include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22,CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signalingmolecule(s) in the CAR contain(s) a cytoplasmic signaling domain,portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the CAR includes a signaling domain and/ortransmembrane portion of a costimulatory receptor, such as CD28, 4-1BB,OX40, DAP10, and ICOS. In some aspects, the same CAR includes both theactivating and costimulatory components.

In some embodiments, the activating domain is included within one CAR,whereas the costimulatory component is provided by another CARrecognizing another antigen. In some embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In some aspects, the cells includeone or more stimulatory or activating CAR and/or a costimulatory CAR. Insome embodiments, the cells further include inhibitory CARs (iCARs, seeFedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such asa CAR recognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta)intracellular domain. In some embodiments, the intracellular signalingdomain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9)co-stimulatory domains, linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more,costimulatory domains and an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. Exemplary CARs include intracellularcomponents of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the CAR or other antigen receptor further includesa marker, such as a cell surface marker, which may be used to confirmtransduction or engineering of the cell to express the receptor, such asa truncated version of a cell surface receptor, such as truncated EGFR(tEGFR). In some aspects, the marker includes all or part (e.g.,truncated form) of CD34, a NGFR, or epidermal growth factor receptor(e.g., tEGFR). In some embodiments, the nucleic acid encoding the markeris operably linked to a polynucleotide encoding for a linker sequence,such as a cleavable linker sequence, e.g., T2A. See WO2014031687.

In some embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof.

In some embodiments, the molecule is a non-self molecule, e.g., non-selfprotein, i.e., one that is not recognized as “self” by the immune systemof the host into which the cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or thirdgeneration CARs. In some aspects, a first generation CAR is one thatsolely provides a CD3-chain induced signal upon antigen binding; in someaspects, a second-generation CARs is one that provides such a signal andcostimulatory signal, such as one including an intracellular signalingdomain from a costimulatory receptor such as CD28 or CD137; in someaspects, a third generation CAR is one that includes multiplecostimulatory domains of different costimulatory receptors.

In some embodiments, the chimeric antigen receptor includes anextracellular portion containing an antibody or antibody fragment. Insome aspects, the chimeric antigen receptor includes an extracellularportion containing the antibody or fragment and an intracellularsignaling domain. In some embodiments, the antibody or fragment includesan scFv and the intracellular domain contains an ITAM. In some aspects,the intracellular signaling domain includes a signaling domain of a zetachain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimericantigen receptor includes a transmembrane domain linking theextracellular domain and the intracellular signaling domain. In someaspects, the transmembrane domain contains a transmembrane portion ofCD28. In some embodiments, the chimeric antigen receptor contains anintracellular domain of a T cell costimulatory molecule. In someaspects, the T cell costimulatory molecule is CD28 or 41BB.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Polypeptides, including the provided receptors and otherpolypeptides, e.g., linkers or peptides, may include amino acid residuesincluding natural and/or non-natural amino acid residues. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, and phosphorylation. In someaspects, the polypeptides may contain modifications with respect to anative or natural sequence, as long as the protein maintains the desiredactivity. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification.

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject generally recognize or specifically bind toa molecule that is expressed in, associated with, and/or specific forthe disease or condition or cells thereof being treated. Upon specificbinding to the molecule, e.g., antigen, the receptor generally deliversan immunostimulatory signal, such as an ITAM-transduced signal, into thecell, thereby promoting an immune response targeted to the disease orcondition. For example, in some embodiments, the cells express a CARthat specifically binds to an antigen expressed by a cell or tissue ofthe disease or condition or associated with the disease or condition.

In some contexts, overexpression of a stimulatory factor (for example, alymphokine or a cytokine) may be toxic to a subject. Thus, in somecontexts, the viral vector introduces into the cell gene segments thatcause the cells to be susceptible to negative selection in vivo, such asupon administration in adoptive immunotherapy. For example, in someaspects, following transduction of the cells with such gene segments,the cells are eliminated as a result of a change in the in vivocondition of the subject to which they are administered. The negativeselectable phenotype may result from the insertion of a gene thatconfers sensitivity to an administered agent, for example, a compound.Negative selectable genes include the Herpes simplex virus type Ithymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977)which confers ganciclovir sensitivity; the cellular hypoxanthinephosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

Among additional nucleic acids that may be included in the viral vectorfor transduction and expression in the cells are those encoding productsthat improve the efficacy of therapy, such as by promoting viabilityand/or function of transferred cells; provide a genetic marker forselection and/or evaluation of the cells, such as to assess in vivosurvival or localization, and/or improve safety, for example, by makingthe cell susceptible to negative selection in vivo as described byLupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell etal., Human Gene Therapy 3:319-338 (1992); see also the publications ofPCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use ofbifunctional selectable fusion genes derived from fusing a dominantpositive selectable marker with a negative selectable marker. See, e.g.,Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

VI. Therapeutic Methods and Compositions

In some aspects, the products of the methods are used in methods oftreatment, e.g., therapeutic methods, such as for administrating thecells and compositions to subjects in adoptive cell therapy. Alsoprovided are such methods and uses of cells processed and produced bythe methods, and pharmaceutical compositions and formulations for usetherein. The provided methods generally involve administering the cellsor compositions, e.g., output composition and/or formulatedcompositions, to subjects.

In some embodiments, the cells express recombinant receptors, such asCARs, or other antigen receptors, such as transgenic TCRs, e.g., thosetransferred in the transduction methods provided herein. Such cellsgenerally are administered to subjects having a disease or conditionspecifically recognized by the receptor. In one embodiment, the cellsexpress a recombinant receptor or a chimeric receptor, such as anantigen receptor, e.g. a CAR or a TCR, that specifically binds to aligand associated with the disease or condition or expressed by a cellor tissue thereof. For example, in some embodiments, the receptor is anantigen receptor and the ligand is an antigen specific for and/orassociated with the disease or condition. The administration generallyeffects an improvement in one or more symptoms of the disease orcondition and/or treats or prevents the disease or condition or asymptom thereof. Among the diseases, conditions, and disorders aretumors, including solid tumors, hematologic malignancies, and melanomas,and including localized and metastatic tumors, infectious diseases, suchas infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV,and parasitic disease, and autoimmune and inflammatory diseases. In someembodiments, the disease or condition is a tumor, cancer, malignancy,neoplasm, or other proliferative disease or disorder. Such diseasesinclude but are not limited to leukemia, lymphoma, e.g., chroniclymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloidleukemia, multiple myeloma, refractory follicular lymphoma, mantle celllymphoma, indolent B cell lymphoma, B cell malignancies, cancers of thecolon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, andbrain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma,pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma,colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma,medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.

In some embodiments, the disease or condition is an infectious diseaseor condition, such as, but not limited to, viral, retroviral, bacterial,and protozoal infections, immunodeficiency, Cytomegalovirus (CMV),Epstein-Ban virus (EBV), adenovirus, BK polyomavirus. In someembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

In some embodiments, antigen associated with the disease or disorderthat is targeted by the cells or compositions is selected from the groupconsisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM,CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen,anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2,EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2,GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, LewisY, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA,NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72,VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen,PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2,CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), acyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/ormolecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the cells or compositions are administered in anamount that is effective to treat or prevent the disease or condition,such as a therapeutically effective or prophylactically effectiveamount. Thus, in some embodiments, the methods of administration includeadministration of the cells and compositions at effective amounts.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and can be determined.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to complete or partial amelioration orreduction of a disease or condition or disorder, or a symptom, adverseeffect or outcome, or phenotype associated therewith. Desirable effectsof treatment include, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.The terms do not imply complete curing of a disease or completeelimination of any symptom or effect(s) on all symptoms or outcomes.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. In some embodiments, the provided cells and compositionsare used to delay development of a disease or to slow the progression ofa disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, cells that suppress tumor growthreduce the rate of growth of the tumor compared to the rate of growth ofthe tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,cells, or composition, in the context of administration, refers to anamount effective, at dosages/amounts and for periods of time necessary,to achieve a desired result, such as a therapeutic or prophylacticresult.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalformulation or cells, refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result, suchas for treatment of a disease, condition, or disorder, and/orpharmacokinetic or pharmacodynamic effect of the treatment. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the subject, and thepopulations of cells administered.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Methods for administration of cells for adoptive cell therapy are knownand may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g.,adoptive T cell therapy, is carried out by autologous transfer, in whichthe cells are isolated and/or otherwise prepared from the subject who isto receive the cell therapy, or from a sample derived from such asubject. Thus, in some aspects, the cells are derived from a subject,e.g., patient, in need of a treatment and the cells, and followingisolation and processing the cells are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by allogeneic transfer, inwhich the cells are isolated and/or otherwise prepared from a subjectother than a subject who is to receive or who ultimately receives thecell therapy, e.g., a first subject. In such embodiments, the cells thenare administered to a different subject, e.g., a second subject, of thesame species. In some embodiments, the first and second subjects aregenetically identical. In some embodiments, the first and secondsubjects are genetically similar. In some embodiments, the secondsubject expresses the same HLA class or supertype as the first subject.

The cells can be administered by any suitable means, for example, bybolus infusion, by injection, e.g., intravenous or subcutaneousinjections, intraocular injection, periocular injection, subretinalinjection, intravitreal injection, trans-septal injection, subscleralinjection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In some embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, a given dose isadministered by a single bolus administration of the cells, by multiplebolus administrations of the cells, or by continuous infusionadministration of the cells.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. The compositionsand cells are in some embodiments suitably administered to the subjectat one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The cells in some embodiments are co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of one or more additional therapeutic agents, or viceversa. In some embodiments, the cells are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellsare administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agents includes a cytokine,such as IL-2, for example, to enhance persistence.

Once the cells are administered to the subject (e.g., human), thebiological activity of the cell populations in some aspects is measuredby any of a number of known methods. Parameters to assess includespecific binding of the cells to antigen, in vivo, e.g., by imaging, orex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, theability of the cells to destroy target cells can be measured using anysuitable method known in the art, such as cytotoxicity assays describedin, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702(2009), and Herman et al. J. Immunological Methods, 285(1): 25-40(2004). In certain embodiments, the biological activity of the cellsalso can be measured by assaying expression and/or secretion of certaincytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects thebiological activity is measured by assessing clinical outcome, such asreduction in tumor burden or load. In some aspects, toxic outcomes,persistence and/or expansion of the cells, and/or presence or absence ofa host immune response, are assessed.

In certain embodiments, the cells are modified in any number of ways,such that their therapeutic or prophylactic efficacy is increased. Forexample, the engineered CAR or TCR expressed by the population can beconjugated either directly or indirectly through a linker to a targetingmoiety. The practice of conjugating compounds, e.g., the CAR or TCR, totargeting moieties is known in the art. See, for instance, Wadwa et al.,J. Drug Targeting 3: 111 (1995), and U.S. Pat. No. 5,087,616.

Also provided are pharmaceutical compositions or formulations for use insuch methods, which in some embodiments are formulated in connectionwith the provided processing methods, such as in the closed system inwhich other processing steps are carried out, such as in an automated orpartially automated fashion.

In some embodiments, the cells and compositions are administered to asubject in the form of a pharmaceutical composition or formulation, suchas a composition comprising the cells or cell populations and apharmaceutically acceptable carrier or excipient.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

The pharmaceutical compositions in some embodiments additionallycomprise other pharmaceutically active agents or drugs, such aschemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc. In some embodiments, the agents are administered inthe form of a salt, e.g., a pharmaceutically acceptable salt. Suitablepharmaceutically acceptable acid addition salts include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric, and sulphuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, and arylsulphonic acids, for example,p-toluenesulphonic acid.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by theparticular cell and/or by the method of administration. Accordingly,there are a variety of suitable formulations. For example, thepharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition. Carriers are described, e.g., byRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being treated with thecells, preferably those with activities complementary to the cells,where the respective activities do not adversely affect one another.Such active ingredients are suitably present in combination in amountsthat are effective for the purpose intended. Thus, in some embodiments,the pharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, and/or vincristine.

The pharmaceutical composition in some embodiments contains the cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. The desired dosage can bedelivered by a single bolus administration of the cells, by multiplebolus administrations of the cells, or by continuous infusionadministration of the cells.

The cells and compositions may be administered using standardadministration techniques, formulations, and/or devices. Administrationof the cells can be autologous or heterologous. For example,immunoresponsive cells or progenitors can be obtained from one subject,and administered to the same subject or a different, compatible subject.Peripheral blood derived immunoresponsive cells or their progeny (e.g.,in vivo, ex vivo or in vitro derived) can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, or parenteraladministration. When administering a therapeutic composition (e.g., apharmaceutical composition containing a genetically modifiedimmunoresponsive cell), it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, the cellpopulations are administered parenterally. The term “parenteral,” asused herein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecells are administered to the subject using peripheral systemic deliveryby intravenous, intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyoi (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can contain auxiliary substancessuch as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, and/or colors, dependingupon the route of administration and the preparation desired. Standardtexts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Among the processing steps may include formulating such compositions.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.” It is understood thataspects and variations described herein include “consisting” and/or“consisting essentially of” aspects and variations.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be constcued as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control under otherwiseidentical conditions and/or at a level substantially similar to that forcell known to be positive for the marker, and/or at a levelsubstantially higher than that for a cell known to be negative for themarker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control underotherwise identical conditions, and/or at a level substantially lowerthan that for cell known to be positive for the marker, and/or at alevel substantially similar as compared to that for a cell known to benegative for the marker.

VII. Exemplary Embodiments

Among the embodiments provided herein are:

1. A transduction method, comprising incubating, in an internal cavityof a centrifugal chamber, an input composition comprising cells andviral particles containing a recombinant viral vector, wherein

said centrifugal chamber is rotatable around an axis of rotation andcomprises an end wall, a substantially rigid side wall extending fromsaid end wall, and at least one opening, at least a portion of said sidewall surrounding said internal cavity and said at least one openingbeing capable of permitting intake of liquid into said internal cavityand expression of liquid from said cavity;

the centrifugal chamber is rotating around said axis of rotation duringat least a portion of the incubation; and

the method generates an output composition comprising a plurality of thecells transduced with the viral vector.

2. A transduction method, comprising incubating, in an internal cavityof a centrifugal chamber, an input composition comprising cells and aviral particle containing a recombinant viral vector,

said centrifugal chamber being rotatable around an axis of rotation andcomprising an end wall, a substantially rigid side wall extending fromsaid end wall, and at least one opening, wherein at least a portion ofsaid side wall surrounds said internal cavity and said at least oneopening is capable of permitting intake of liquid into said internalcavity and expression of liquid from said cavity, wherein:

the centrifugal chamber is rotating around the axis of rotation duringat least a portion of the incubation;

the total liquid volume of said input composition present in said cavityduring rotation of said centrifugal chamber is no more than about 5 mLper square inch of the internal surface area of the cavity; and

the method generates an output composition comprising a plurality of thecells transduced with the viral vector.

3. The method of embodiment 1 or embodiment 2, wherein said rotatingcomprises rotation at a relative centrifugal force (RCF) at an internalsurface of the side wall of the cavity and/or at a surface layer of thecells of greater than at or about 200 g, greater than at or about 300 g,or greater than at or about 500 g.

4. The method of any of embodiments 1-3, wherein said rotating comprisesrotation at a relative centrifugal force at an internal surface of theside wall of the cavity and/or at a surface layer of the cells that is:

at or about 600 g, 800 g, 1000 g, 1100 g, 1600 g, 2000 g, 2100 g, 2200g, 2500 g or 3000 g; or

at least at or about 600 g, 800 g, 1000 g, 1100 g, 1600 g, 2000 g, 2100g, 2200 g, 2500 g or 3000 g.

5. The method of any of embodiments 1-4, wherein said rotating comprisesrotation at a relative centrifugal force at an internal surface of theside wall of the cavity and/or at a surface layer of the cells that isbetween or between about 500 g and 2500 g, 500 g and 2000 g 500 g and1600 g, 500 g an 1000 g, 600 g and 1600 g, 600 g and 1000 g, 1000 g and2000 g or 1000 g and 1600 g, each inclusive.

6. The method of any of embodiments 1-5, wherein the at least a portionof the incubation during which the chamber is rotating is for a timethat is:

greater than or about 5 minutes, greater than or about 10 minutes,greater than or about 15 minutes, greater than or about 20 minutes,greater than or about 30 minutes, greater than or about 45 minutes,greater than or about 60 minutes, greater than or about 90 minutes orgreater than or about 120 minutes; or

between or between about 5 minutes and 60 minutes, 10 minutes and 60minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30minutes and 60 minutes or 45 minutes and 60 minutes, each inclusive.

7. The transduction method of any of embodiments 1-6, wherein saidcentrifugal chamber further comprises a movable member and said internalcavity is a cavity of variable volume defined by said end wall, saidsubstantially rigid side wall, and said movable member, said movablemember being capable of moving within the chamber to vary the internalvolume of the cavity.

8. The method of any of embodiments 1-7, wherein said side wall iscurvilinear.

9. The method of embodiment 8, wherein said side wall is generallycylindrical.

10. The method of any of embodiments 7-9, wherein:

the movable member is a piston; and/or

the movable member is capable of axially moving within the chamber tovary the internal volume of the cavity.

11. The method of any of embodiments 1-10, wherein

said at least one opening comprises an inlet and an outlet, respectivelycapable of permitting said intake and expression; or

said at least one opening comprises a single inlet/outlet, capable ofpermitting said intake and said expression.

12. The method of any of embodiments 1-11, wherein said at least oneopening is coaxial with the chamber and is located in the end wall.

13. The method of any of embodiments 1-12, wherein:

the internal surface area of said cavity is at least at or about 1×10⁹μm²;

the internal surface area of said cavity is at least at or about 1×10¹⁰μm²;

the length of said rigid wall in the direction extending from said endwall is at least about 5 cm;

the length of said rigid wall in the direction extending from said endwall is at least about 8 cm; and/or

the cavity comprises a radius of at least about 2 cm at least onecross-section.

14. The method of any of embodiments 1-13, wherein:

the average liquid volume of said input composition present in saidcavity during said incubation is no more than about 5 milliliters (mL)per square inch of the internal surface area of the cavity during saidincubation;

the maximum liquid volume of said input composition present in saidcavity at any one time during said incubation is no more than about 5 mLper square inch of the maximum internal surface area of the cavity;

the average liquid volume of said input composition present in saidcavity during said incubation is no more than about 2.5 milliliters (mL)per square inch of the internal surface area of the cavity during saidincubation; or

the maximum liquid volume of said input composition present in saidcavity at any one time during said incubation is no more than about 2.5mL per square inch of the maximum internal surface area of the cavity.

15. The method of any of embodiments 1-14, wherein the liquid volume ofsaid input composition present in said cavity during said rotation isbetween or between about 0.5 mL per square inch of the internal surfacearea of the cavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5mL/sq.in., 0.5 mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1mL/sq.in. and 2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in.

16. The method of any of embodiments 1-15, wherein:

the number of said cells in said input composition is at or about thenumber of said cells sufficient to form a monolayer on the surface ofsaid cavity during rotation of said centrifugal chamber at a force of ator about 1000 g or at or about 2000 g at an internal surface of the sidewall and/or at a surface layer of the cells; and/or

the number of said cells in said input composition is no more than 1.5times or 2 times the number of said cells sufficient to form a monolayeron the surface of said cavity during rotation of said centrifugalchamber at a force of at or about 1000 g or at or about 2000 g at aninternal surface of the side wall and/or at a surface layer of thecells.

17. The method of any of embodiments 1-16, wherein

said input composition in the cavity comprises at least at or about1×10⁶ of said cells; or

said input composition in the cavity comprises at least at or about5×10⁶ of said cells; or

said input composition in the cavity comprises at least at or about1×10⁷ of said cells; or

said input composition in the cavity comprises at least at or about1×10⁸ of said cells.

18. The method of any of embodiments 1-17, wherein said inputcomposition in the cavity comprises at least at or about 1×10⁷ of saidcells, at least at or about 2×10⁷ of said cells, at least at or about3×10⁷ of said cells, at least at or about 4×10⁷ of said cells, at leastat or about 5×10⁷ of said cells, at least at or about 6×10⁷ of saidcells, at least at or about 7×10⁷ of said cells, at least at or about8×10⁷ of said cells, at least at or about 9×10⁷ of said cells, at leastat or about 1×10⁸ of said cells, at least at or about 2×10⁸ of saidcells, at least at or about 3×10⁸ of said cells or at least at or about4×10⁸ of said cells.

19. The method of any of embodiments 1-18, wherein:

said input composition comprises at least at or about 1 infectious unit(IU) of viral particles per one of said cells, at least at or about 2 IUper one of said cells, at least at or about 3 IU per one of said cells,at least at or about 4 IU per one of said cells, at least at or about 5IU per one of said cells, at least at or about 10 IU per one of saidcells, at least at or about 20 IU per one of said cells, at least at orabout 30 IU per one of said cells, at least at or about 40 IU per one ofsaid cells, at least at or about 50 IU per one of said cells, or atleast at or about 60 IU per one of said cells; or

said input composition comprises at or about 1 infectious unit (IU) ofviral particles per one of said cells, at or about 2 IU per one of saidcells, at or about 3 IU per one of said cells, at or about 4 IU per oneof said cells, at or about 5 IU per one of said cells, at or about 10 IUper one of said cells, at or about 20 IU per one of said cells, at orabout 30 IU per one of said cells, at or about 40 IU per one of saidcells, at or about 50 IU per one of said cells, or at or about 60 IU perone of said cells.

20. The method of any of embodiments 1-19, wherein:

the maximum total liquid volume of said input composition present insaid cavity at any one time during said incubation is no more than 2times, no more than 10 times, or no more than 100 times, the totalvolume of said cells in said cavity or the average volume of the inputcomposition over the course of the incubation is no more than 2, 10, or100 times the total volume of cells in the cavity.

21. The method of any of embodiments 1-20, wherein the maximum volume ofsaid input composition present in said cavity at any one time duringsaid incubation or the average volume over the course of the incubationis no more than at or about 2 times, 10 times, 25 times, 50 times, 100times, 500 times, or 1000 times the volume of a monolayer of said cellsformed on the inner surface of said cavity during rotation of saidchamber at a force of at or about 1000 g or at or about 2000 g at aninternal surface of the side wall and/or at a surface layer of thecells.

22. The method of any of embodiments 1-21, wherein the liquid volume ofthe input composition is no more than 20 mL, no more than 40 mL, no morethan 50 mL, no more than 70 mL, no more than 100 mL, no more than 120mL, no more than 150 mL or no more than 200 mL.

23. The method of any of embodiments 1-22, wherein the input compositionoccupies all or substantially all of the volume of the internal cavityduring at least a portion of said incubation.

24. The method of any of embodiments 1-23, wherein, during at least aportion of the incubation in the chamber or during the rotation of thechamber, the liquid volume of the input composition occupies only aportion of the volume of the internal cavity of the chamber, the volumeof the cavity during said at least a portion or during said rotationfurther comprising a gas, said gas taken into said cavity via said atleast one opening, prior to or during said incubation.

25. The method of embodiment 24, wherein the centrifugal chambercomprises a movable member, whereby intake of gas into the centrifugalchamber effects movement of the movable member to increase the volume ofthe internal cavity of the chamber, thereby decreasing the total liquidvolume of said input composition present in said cavity during rotationof said centrifugal chamber per square inch of the internal surface areaof the cavity compared to the absence of gas in the chamber.

26. A method of transduction, comprising:

a) providing to an internal cavity of a centrifugal chamber that has aninternal surface area of at least at or about 1×10⁹ μm² or at least ator about 1×10¹⁰ μm²:

-   -   i) an input composition comprising cells and viral particles        comprising a recombinant viral vector, wherein:        -   the number of cells in the input composition is at least            1×10⁷ cells, and        -   the viral particles are present in the input composition at            least at or about 1 infectious unit (IU) per one of said            cells, and        -   the input composition comprises a liquid volume that is less            than the maximum volume of the internal cavity of the            centrifugal chamber; and    -   ii) gas at a volume that is up to the remainder of the maximum        volume of the internal cavity of the centrifugal chamber; and

b) incubating the input composition, wherein at least a portion of theincubation is carried out in said internal cavity of said centrifugalchamber while effecting rotation of said centrifugal chamber; and

wherein the method generates an output composition comprising aplurality of the cells transduced with the viral vector.

27. The method of embodiment 26, wherein:

the number of cells is at least at or about 50×10⁶ cells; 100×10⁶ cells;or 200×10⁶ cells; and/or

the viral particles are present at least 1.6 IU/cell, 1.8 IU/cell, 2.0IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell or 3.6 IU/cell, 4.0IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cellor 10.0 IU/cell.

28. The method of embodiment 26 or embodiment 27, wherein:

the liquid volume of the input composition is less than or equal to 200mL, less than or equal to 100 mL, less than or equal to 50 mL or lessthan or equal to 20 mL; and/or

the liquid volume of the input composition is no more than 50%, no morethan 40%, no more than 30%, no more than 20%, or no more than 10% of thevolume of the internal surface area of the cavity during rotation or themaximum internal surface area of the cavity.

29. The method of any of embodiments 26-28, wherein the volume of gas isup to 200 mL, up to 180 mL, up to 140 mL or up to 100 mL.

30. The method of any of embodiments 26-29, wherein said rotation is ata relative centrifugal force at an internal surface of the side wall ofthe cavity or at a surface layer of the cells of at least at or about600 g, 800 g, 1000 g, 1100 g, 1500 g, 1600 g, 2000 g, 2400 g, 2600 g,2800 g, 3000 g, 3200 g or 3600 g.

31. A method of transduction, comprising incubating an input compositioncomprising cells and viral particles comprising a recombinant viralvector, at least a portion of said incubating being carried out underrotating conditions, thereby generating an output composition comprisinga plurality of the cells transduced with the viral vector, wherein:

said input composition comprises greater than or about 20 mL, 50 mL, atleast 100 mL, or at least 150 mL in volume, and/or said inputcomposition comprises at least 1×10⁸ cells; and

said rotating conditions comprise a relative centrifugal force on asurface layer of the cells of greater than about 800 g or greater thanabout 1000 g or greater than about 1500 g.

32. The method of embodiment 31, wherein:

at least 25% or at least 50% of said cells in the output composition aretransduced with said viral vector; and/or

at least 25% or at least 50% of said cells in the output compositionexpress a product of a heterologous nucleic acid comprised within saidviral vector.

33. The method of embodiment 31 or embodiment 32, wherein saidincubation is carried out in a cavity of a centrifugal chamber and thenumber of said cells in said input composition is at or about the numberof said cells sufficient to form a monolayer or a bilayer on the innersurface of said cavity during said rotation.

34. The method of embodiment 33, wherein said centrifugal chambercomprises an end wall, a substantially rigid side wall extending fromsaid end wall, and at least one opening, wherein at least a portion ofsaid side wall surrounds said internal cavity and said at least oneopening is capable of permitting intake of fluid into said internalcavity and expression of fluid from said cavity.

35. The method of embodiment 34, wherein said centrifugal chamberfurther comprises a movable member and said internal cavity is a cavityof variable volume defined by said end wall, said substantially rigidside wall, and said movable member, said movable member being capable ofmoving within the chamber to vary the internal volume of the cavity.

36. The method of any of embodiments 1-30 or 33-35, wherein the inputcomposition in said cavity comprises a liquid volume of at least 20 mLor at least 50 mL and at or about 1 million cells per cm² of theinternal surface area of the cavity during at least a portion of saidincubation.

37. The method of any of embodiments 1-36, wherein a further portion ofthe incubation is carried out outside of the centrifugal chamber and/orwithout rotation, said further portion carried out subsequent to the atleast a portion carried out in the chamber and/or with rotation.

38. The method of any of embodiments 1-37, wherein the at least aportion of the incubation carried out in the cavity of the centrifugalchamber and/or the further portion of the incubation is effected at orat about 37° C.±2° C.

39. The method of embodiment 37 or embodiment 38, wherein the incubationfurther comprises transferring at least a plurality of the cells to acontainer during said incubation and said further portion of theincubation is effected in the container.

40. The method of embodiment 39, wherein the transferring is performedwithin a closed system, wherein the centrifugal chamber and containerare integral to the closed system.

41. The method of any of embodiments 37-40, wherein:

the incubation is carried out for a time between at or about 1 hour andat or about 96 hours, between at or about 4 hours and at or about 72hours, between at or about 8 hours and at or about 48 hours, between ator about 12 hours and at or about 36 hours, between at or about 6 hoursand at or about 24 hours, between at or about 36 hours and at or about96 hours, inclusive; or

the further portion of the incubation is carried out for a time betweenat or about 1 hour and at or about 96 hours, between at or about 4 hoursand at or about 72 hours, between at or about 8 hours and at or about 48hours, between at or about 12 hours and at or about 36 hours, between ator about 6 hours and at or about 24 hours, between at or about 36 hoursand at or about 96 hours, inclusive.

42. The method of any of embodiments 37-41, wherein:

the incubation is carried out for a time that is no more than 48 hours,no more than 36 hours or no more than 24 hours; or

the further portion of the incubation is carried out for a time that isno more than 48 hours, no more than 36 hours or no more than 24 hours.

43. The method of any of embodiments 37-41, wherein:

the incubation is performed in the presence of a stimulating agent;and/or

the further portion of the incubation is performed in the presence of astimulating agent.

44. The method of any of embodiments 37-41, wherein:

the incubation is carried out for a time that is no more than 24 hours;

the cells in the composition have not been subjected to a temperature ofgreater than 30° C. for more than 24 hours; and/or

the incubation is not performed in the presence of a stimulating agent.

45. The method of embodiment 43 or embodiment 44, wherein thestimulating agent is an agent capable of inducing proliferation of Tcells, CD4+ T cells and/or CD8+ T cells.

46. The method of any of embodiments 43-45, wherein the stimulatingagent is a cytokine selected from among IL-2, IL-15 and IL-7.

47. The method of any of embodiments 1-46, wherein the outputcomposition containing transduced cells comprises at least at or about1×10⁷ cells or at least at or about 5×10⁷ cells.

48. The method of embodiment 47, wherein the output compositioncontaining transduced cells comprises at least at or about 1×10⁸ cells,2×10⁸ cells, 4×10⁸ cells, 6×10⁸, 8×10⁸ cells or 1×10⁹ cells.

49. The method of embodiment 47 or embodiment 48, wherein the cells areT cells.

50. The method of embodiment 49, wherein the T cells are unfractionatedT cells, isolated CD4+ T cells and/or isolated CD8+ T cells.

51. The method of any of embodiments 1-50, wherein the method results inintegration of the viral vector into a host genome of one or more of theat least a plurality of cells and/or into a host genome of at least ator about 20% or at least at or about 30% or at least at or about 40% ofthe cells in the output composition.

52. The method of any of embodiments 1-51, wherein:

at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 75% of said cells in said input composition are transduced withsaid viral vector by the method; and/or

at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 75% of said cells in said output composition are transduced withsaid viral vector; and/or

at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 75% of said cells in said output composition express a product ofa heterologous nucleic acid comprised within said viral vector.

53. The method of any of embodiments 1-52, wherein, for an inputcomposition comprising a virus at a ratio of about 1 or about 2 IU percells, said method is capable of producing an output composition inwhich at least 10%, at least 25%, at least 30%, at least 40%, at least50%, or at least 75% of the cells in said output composition generatedby the method comprise said recombinant viral vector and/or express aproduct of a recombinant nucleic acid comprised within said vector.

54. The method of any of embodiments 1-53, wherein:

among all the cells in said output composition that contain therecombinant viral vector or into which the viral vector is integrated,the average copy number of said recombinant viral vector is no more thanabout 10, no more than about 5, no more than about 2.5, or no more thanabout 1.5; or

among the cells in the output composition, the average copy number ofsaid vector is no more than about 2, no more than about 1.5, or no morethan about 1.

55. The method of any of embodiments 1-30 and 33-54, wherein thecentrifugal chamber is integral to a closed system, said closed systemcomprising said chamber and at least one tubing line operably linked tothe at least one opening via at least one connector, whereby liquid andgas are permitted to move between said cavity and said at least onetubing line in at least one configuration of said system.

56. The method of embodiment 55, wherein:

said at least one tubing line comprises a series of tubing lines;

said at least one connector comprises a plurality of connectors; and

said closed system further comprises at least one container operablylinked to said series of tubing lines, the connection permitting liquidand/or gas to pass between said at least one container and said at leastone opening via the series of tubing lines.

57. The method of embodiment 55 or 56, wherein said at least oneconnector comprises a connector selected from the group consisting ofvalves, luer ports, and spikes.

58. The method of any of embodiments 55-57, wherein said at least oneconnector comprises a rotational valve.

59. The method of embodiment 58, wherein said rotational valve is astopcock or multirotational port.

60. The method of any of embodiments 55-59, wherein said at least oneconnector comprises an aseptic connector.

61. The method of any of embodiments 56-60, wherein said at least onecontainer comprises a container selected from the group consisting ofbags, vials, and syringes.

62. The method of any of embodiments 56-61, wherein said at least onecontainer comprises a diluent container, a waste container, a productcollection container, and/or an input product container.

63. The method of any of embodiments 56-62, wherein:

said at least one container comprises at least one input containercomprising said viral vector particles and said cells, a wastecontainer, a product container, and at least one diluent container, eachconnected to said cavity via said series of tubing lines and said atleast one opening.

64. The method of embodiment 63, wherein said method further comprises,prior to and/or during said incubation, effecting intake of said inputcomposition into said cavity, said intake comprising flowing of liquidfrom said at least one input container into said cavity through said atleast one opening.

65. The method of any of embodiments 56-64, wherein at least onecontainer further comprises a container that comprises a gas prior toand/or during at least a point during said incubation and/or the closedsystem further comprises a microbial filter capable of taking in gas tothe internal cavity of the centrifugal chamber and/or the closed systemcontains a syringe port for effecting intake of gas.

66. The method of embodiment 65, wherein the method comprises, prior toand/or during said incubation, providing or effecting intake of gas intosaid cavity under sterile conditions, said intake being effected by (a)flow of gas from the container that comprises gas, (b) flow of gas froman environment external to the closed system, via the microbial filter,or (c) flow of gas from a syringe connected to the system at the syringeport.

67. The method of embodiment 66, wherein the effecting intake of the gasinto the internal cavity of the centrifugal chamber is carried outsimultaneously or together with the effecting intake of the inputcomposition to the internal cavity of the centrifugal chamber.

68. The method of embodiment 66 or embodiment 67, wherein the inputcomposition and gas are combined in a single container under sterileconditions outside of the chamber prior to said intake of said inputcomposition and gas into the internal cavity of the centrifugal chamber.

69. The method of embodiment 68, wherein the effecting of the intake ofthe gas is carried out separately, either simultaneously orsequentially, from the effecting of the intake of the input compositioninto said cavity.

70. The method of any of embodiments 66-69, wherein the intake of gas iseffected by permitting or causing flow of the gas from a sterile closedcontainer comprising the gas, an external environment through amicrobial filter, or a syringe comprising said gas.

71. The method of any of embodiments 24-70, wherein the gas is air.

72. The method of any of embodiments 1-71, wherein the incubation ispart of a continuous process, the method further comprising:

during at least a portion of said incubation, effecting continuousintake of said input composition into said cavity during rotation of thechamber; and

during a portion of said incubation, effecting continuous expression ofliquid from said cavity through said at least one opening duringrotation of the chamber.

73. The method of embodiment 72, further comprising:

during a portion of said incubation, effecting continuous intake of gasinto said cavity during rotation of the chamber; and/or

during a portion of said incubation, effecting continuous expression ofgas from said cavity.

74. The method of embodiment 73, wherein the method comprises theexpression of liquid and the expression of gas from said cavity, whereeach is expressed, simultaneously or sequentially, into a differentcontainer.

75. The method of any of embodiments 72-74, wherein at least a portionof the continuous intake and the continuous expression occursimultaneously.

76. The method of any of embodiments 1-75, wherein the incubation ispart of a semi-continuous process, the method further comprising:

prior to said incubation, effecting intake of said input composition,and optionally gas, into said cavity through said at least one opening;

subsequent to said incubation, effecting expression of liquid and/oroptionally gas from said cavity;

effecting intake of another input composition comprising cells and saidviral particles containing a recombinant viral vector, and optionallygas, into said internal cavity; and

incubating said another input composition in said internal cavity,

wherein the method generates another output composition comprising aplurality of cells of the another input composition that are transducedwith said viral vector.

77. The method of any of embodiments 64-76, wherein said providing orsaid intake of the input composition into the cavity comprises:

intake of a single composition comprising the cells and the viralparticles containing the recombinant viral vector; or

intake of a composition comprising the cells and a separate compositioncomprising the viral particles containing the recombinant viral vector,whereby the compositions are mixed, effecting intake of the inputcomposition.

78. The method of embodiment 64-77, wherein the method furthercomprises:

effecting rotation of said centrifugal chamber prior to and/or duringsaid incubation;

effecting expression of liquid from said cavity into said wastecontainer following said incubation;

effecting expression of liquid from said at least one diluent containerinto said cavity via said at least one opening and effecting mixing ofthe contents of said cavity; and

effecting expression of liquid from said cavity into said productcontainer, thereby transferring cells transduced with the viral vectorinto said product container.

79. The method of any of embodiments 1-78, further comprising:

(a) washing a biological sample comprising said cells in an internalcavity of a centrifugal chamber prior to said incubation; and/or

(b) isolating said cells from a biological sample, wherein at least aportion of the isolation step is performed in an internal cavity of acentrifugal chamber prior to said incubation; and/or

(c) stimulating cells prior to and/or during said incubation, saidstimulating comprising exposing said cells to stimulating conditions,thereby inducing cells of the input composition to proliferate, whereinat least a portion of the step of stimulating cells is performed in aninternal cavity of a centrifugal chamber.

80. The method of embodiment 79, wherein said isolating comprisescarrying out immunoaffinity-based selection.

81. The method of embodiment 79 or 80, wherein said stimulatingconditions comprise the presence of an agent capable of activating oneor more intracellular signaling domains of one or more components of aTCR complex.

82. The method of embodiment 81, wherein said agent comprises a primaryagent that specifically binds to a member of a TCR complex and asecondary agent that specifically binds to a T cell costimulatorymolecule.

83. The method of embodiment 82, wherein the primary agent specificallybinds to CD3; and/or

the costimulatory molecule is selected from the group consisting ofCD28, CD137 (4-1-BB), OX40, or ICOS.

84. The method of embodiment 83, wherein said primary and secondaryagents comprise antibodies and/or are present on the surface of a solidsupport.

85. The method of any of embodiments 79-84, wherein said biologicalsample in (a) and/or in (b) is or comprises a whole blood sample, abuffy coat sample, a peripheral blood mononuclear cells (PBMC) sample,an unfractionated T cell sample, a lymphocyte sample, a white blood cellsample, an apheresis product, or a leukapheresis product.

86. The method of any of embodiments 1-85, further comprisingformulating cells transduced by the method in a pharmaceuticallyacceptable buffer in an internal cavity of a centrifugal chamber,thereby producing a formulated composition.

87. The method of embodiment 86, further comprising effecting expressionof the formulated composition to one or a plurality of containers.

88. The method of embodiment 87, wherein the effecting of expression ofthe formulated composition comprises effecting expression of a number ofthe cells present in a single unit dose to one or each of said one or aplurality of containers.

89. The method of any of embodiments 79-88, wherein each of said acavity of a centrifugal chamber is the same or different as a cavity ofa centrifugal employed in one or more of the other steps and/or in theprocess of incubating and/or rotating an input composition containingcells and viral particles.

90. The method of any of embodiments 79-89, wherein each of saidcentrifugal chambers is integral to a closed system, said closed systemcomprising said chamber and at least one tubing line operably linked tothe at least one opening via at least one connector, whereby liquid andgas are permitted to move between said cavity and said at least onetubing line in at least one configuration of said system.

91. The method of any of embodiments 1-90, wherein said cells in saidinput composition are primary cells.

92. The method of any of embodiments 1-91, wherein:

said cells in said input composition comprise suspension cells;

said cells in said input composition comprise white blood cells; and/or

said cells in said input composition comprise T cells or NK cells.

93. The method of any of embodiments 1-92, wherein said cells in saidinput composition are unfractionated T cells, isolated CD8⁺ T cells, orisolated CD4⁺ T cells.

94. The method of any of embodiments 1-93, wherein said cells in saidinput composition are human cells.

95. The method of any of embodiments 7-94, wherein, during saidincubation, said centrifugal chamber is associated with a sensor, saidsensor capable of monitoring the position of said movable member, andcontrol circuitry, said circuitry capable of receiving and transmittinginformation to and from said sensor and causing movement of said movablemember, said control circuitry further associated with a centrifugecapable of causing rotation of said chamber during said incubation.

96. The method of any of embodiments 7-95, wherein said chambercomprises said movable member and during said incubation, saidcentrifugal chamber is located within a centrifuge and associated with asensor, said sensor capable of monitoring the position of said movablemember, and control circuitry capable of receiving and transmittinginformation from said sensor and causing movement of said movablemember, intake and expression of liquid and/or gas to and from saidcavity via said one or more tubing lines, and rotation of said chambervia said centrifuge.

97. The method of embodiment 95 or embodiment 96, wherein said chamber,said control circuitry, said centrifuge, and said sensor are housedwithin a cabinet during said incubation.

98. The method of any of embodiments 1-97, wherein said recombinantviral vector encodes a recombinant receptor, which is thereby expressedby cells of the output composition.

99. The method of embodiment 98, wherein said recombinant receptor is arecombinant antigen receptor.

100. The method of embodiment 99, wherein said recombinant antigenreceptor is a functional non-T cell receptor.

101. The method of embodiment 100, wherein said functional non-T cellreceptor is a chimeric antigen receptor (CAR).

102. The method of embodiment 99, wherein said recombinant antigenreceptor is a transgenic T cell receptor (TCR).

103. The method of embodiment 99, wherein said recombinant receptor is achimeric receptor comprising an extracellular portion that specificallybinds to a ligand and an intracellular signaling portion containing anactivating domain and a costimulatory domain.

104. The method of any of embodiments 1-103, wherein:

the cells comprise primary human T cells obtained from a human subject;and

prior to said incubation and/or prior to completion of said transductionand/or, where the method includes formulation, prior to the formulation,the primary human T cells have not been present externally to thesubject at a temperature of greater than 30° C. for greater than 1 hour,greater than 6 hours, greater than 24 hours, or greater than 48 hours;or

prior to said incubation and/or prior to the completion of thetransduction, and/or where the method includes formulation, prior to theformulation, the primary human T cells have not been incubated in thepresence of an antibody specific for CD3 and/or an antibody specific forCD28 and/or a cytokine, for greater than 1 hour, greater than 6 hours,greater than 24 hours, or greater than 48 hours.

105. A method for selection, the method comprising:

(a) incubating a selection reagent and primary cells in an internalcavity of a centrifugal chamber under mixing conditions, whereby aplurality of the primary cells bind to said selection reagent; and

(b) separating said plurality of said primary cells from another one ormore of the primary cells based on binding to the selection reagent,

thereby enriching the primary cells based on binding to the selectionreagent,

wherein said centrifugal chamber is rotatable around an axis of rotationand said internal cavity has a maximum volume of at least 50, at least100, or at least 200 mL.

106. A method for stimulation of cells, the method comprising incubatinga stimulation agent and primary cells under conditions whereby thestimulation agent binds to a molecule expressed by a plurality of theprimary cells and said plurality of the cells are activated orstimulated, wherein

at least a portion of the incubation being carried out in an internalcavity of a centrifugal chamber under mixing conditions,

said centrifugal chamber is rotatable around an axis of rotation; and

said internal cavity has a maximum volume of at least 50, at least 100,or at least 200 mL.

107. The method of embodiment 105 or embodiment 106, wherein the chamberfurther comprises an end wall, a substantially rigid side wall extendingfrom said end wall, and at least one opening, wherein at least a portionof said side wall surrounds said internal cavity and said at least oneopening is capable of permitting intake of liquid into said internalcavity and expression of liquid from said cavity.

108. A composition, comprising transduced cells produced by the methodof any of embodiments 1-107.

109. The composition of embodiment 108, wherein said cells:

are primary cells; and/or

are human cells; and/or

comprise white blood cells; and/or

comprise T cells; and/or

comprise NK cells.

110. The composition of embodiment 108 or embodiment 109, wherein thecomposition comprises at least at or about 5×10⁷ cells, 1×10⁸ cells,2×10⁸ cells, 4×10⁸ cells, 6×10⁸, 8×10⁸ cells or 1×10⁹ cells.

111. The composition of any of embodiments 106-110, wherein thecomposition comprises a therapeutically effective number of cells foruse in adoptive T cell therapy.

112. The composition of any of embodiments 106-111, wherein:

the cells are T cells; and

subsequent to transduction, the cells in the composition are notsubjected to cell expansion in the presence of a stimulating agentand/or the cells are not incubated at a temperature greater than 30° C.for more than 24 hours or the composition does not contain a cytokine orthe composition does not contain a stimulating agent that specificallybinds to CD3 or a TCR complex.

113. A composition, comprising at least 1×10⁷ or at least 5×10⁷ cells Tcells, at least a plurality of which are transduced with a recombinantviral vector or express a recombinant or engineered antigen receptor,wherein:

subsequent to transduction, the cells in the composition have not beensubjected to cell expansion in the presence of a stimulating agent;and/or

subsequent to transduction, the cells have not been incubated at atemperature greater than 30° C. for more than 24 hours.

114. A composition, comprising at least 1×10⁷ or at least 5×10⁷ primaryhuman T cells, at least a plurality of which are transduced with arecombinant viral vector or express a recombinant or engineered antigenreceptor, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Tcells in the composition comprise high expression of CD69 and/orTGF-beta-II.

115. The composition of embodiment 114, wherein said at least 30%, 40%,50%, 60%, 70%, 80%, or 90% of the T cells in the composition comprise nosurface expression of CD62L and/or comprise high expression of CD25,ICAM, GM-CSF, IL-8 and/or IL-2.

116. The composition of any of embodiments 113-115, wherein saidcomposition comprises at least 1×10⁸ cells, 2×10⁸ cells, 4×10⁸ cells,6×10⁸, 8×10⁸ cells or 1×10⁹ cells.

117. The composition of any of embodiments 109-116, wherein said T cellsare unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ Tcells.

118. The composition of any of embodiments 108-117, wherein at least2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, or at least 75%of said cells in said composition are transduced with the viral vector.

119. The composition of any of embodiments 108-118, wherein:

the viral vector encodes a recombinant receptor; and

transduced cells in the composition express the recombinant receptor.

120. The composition of embodiment 119, wherein said recombinantreceptor is a recombinant antigen receptor.

121. The composition of embodiment 120, wherein said recombinant antigenreceptor is a functional non-T cell receptor.

122. The composition of embodiment 121, wherein said functional non-Tcell receptor is a chimeric antigen receptor (CAR).

123. The composition of any of embodiments 119-122, wherein saidrecombinant receptor is a chimeric receptor comprising an extracellularportion that specifically binds to a ligand and an intracellularsignaling portion containing an activating domain and a costimulatorydomain.

124. The composition of embodiment 120, wherein said recombinant antigenreceptor is a transgenic T cell receptor (TCR).

125. The composition of any of embodiments 110-124, wherein:

among all the cells in the composition, the average copy number of saidrecombinant viral vector is no more than about 10, no more than 8, nomore than 6, no more than 4, or no more than about 2; or

among the cells in the composition transduced with the recombinant viralvector, the average copy number of said vector is no more than about 10,no more than 8, no more than 6, no more than 4, or no more than about 2.

126. The composition of any of embodiments 110-125, comprising apharmaceutically acceptable excipient.

127. An article of manufacture comprising a container or plurality ofcontainers, the container or the plurality of containers collectivelycontaining a composition according to any of embodiments 113-126.

128. The article of manufacture of embodiment 127, wherein the containeror plurality of containers comprises two or more or three or more bagsand the composition further comprises a pharmaceutically acceptableexcipient.

129. A method of treatment, the method comprising administering to asubject having a disease or condition the composition of any ofembodiments 110-126.

130. The method of embodiment 129, wherein the transduced T cells in thecomposition exhibit increased or longer expansion and/or persistence inthe subject than transduced T cells in a composition in which,subsequent to transduction, the cells in the composition have beensubjected to cell expansion in the presence of a stimulating agentand/or the cells have been incubated at a temperature greater than 30°C. for more than 24 hours.

131. The method of embodiment 129 or embodiment 130, wherein therecombinant receptor, chimeric antigen receptor or transgenic TCRspecifically binds to an antigen associated with the disease orcondition.

132. The method of any of embodiments 129-131, wherein the disease orcondition is a cancer, and autoimmune disease or disorder, or aninfectious disease.

133. A composition, comprising:

-   -   at least 1×10⁷ cells; and

at least at or about 1 infectious unit (IU) per cell of viral particlescomprising a recombinant viral vector.

134. The composition of embodiment 133, wherein:

said cells comprise at least or about 50×10⁶ cells; 100×10⁶ cells; or200×10⁶ cells; and/or

said viral particles are present in the composition in an amount that isat least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.

135. The composition of embodiment 133 or embodiment 134, wherein theliquid volume of the composition is less than or equal to 220 mL, lessthan or equal to 200 mL, less than or equal to 100 mL, less than orequal to 50 mL or less than or equal to 20 mL.

136. The composition of any of embodiments 133-135, wherein said cellsare primary cells.

137. The composition of any of embodiments 133-136, wherein said cellsare human cells.

138. The composition of any of embodiments 133-137, wherein:

said cells comprise suspension cells;

said cells comprise white blood cells; and/or

said cells comprise T cells or NK cells.

139. The composition of embodiment 138, wherein said cells are T cellsand the T cells are unfractionated T cells, isolated CD8+ T cells, orisolated CD4+ T cells.

140. The composition of any of embodiments 133-139, wherein the viralvector encodes a recombinant receptor.

141. The composition of embodiment 140, wherein said recombinantreceptor is a recombinant antigen receptor.

142. The composition of embodiment 141, wherein said recombinant antigenreceptor is a functional non-T cell receptor.

143. The composition of embodiment 142, wherein said functional non-Tcell receptor is a chimeric antigen receptor (CAR).

144. The composition of any of embodiments 140-143, wherein saidrecombinant receptor is a chimeric receptor comprising an extracellularportion that specifically binds to a ligand and an intracellularsignaling portion containing an activating domain and a costimulatorydomain.

145. The composition of embodiment 141, wherein said recombinant antigenreceptor is a transgenic T cell receptor (TCR).

146. A centrifugal chamber rotatable around an axis of rotation, saidchamber comprising an internal cavity comprising the composition of anyof embodiments 110-126.

147. A centrifugal chamber rotatable around an axis of rotation, saidchamber comprising an internal cavity comprising: (a) a compositioncontaining at least 5×10⁷ primary T cells transduced with a recombinantviral vector and/or (b) a composition containing at least 5×10⁷ primaryT cells and viral particles containing a recombinant viral vector.

148. The centrifugal chamber of embodiment 146 or 147, said chamberfurther comprising an end wall, a substantially rigid side wallextending from said end wall, and at least one opening, wherein at leasta portion of said side wall surrounds said internal cavity and said atleast one opening is capable of permitting intake of liquid into saidinternal cavity and expression of liquid from said cavity.

149. The centrifugal chamber of embodiment 147 or 148, wherein saidcomposition in said cavity comprises at least 1×10⁸ cells, 2×10⁸ cells,4×10⁸ cells, 6×10⁸, 8×10⁸ cells or 1×10⁹ of the cells.

150. The centrifugal chamber of embodiment 147 or embodiment 148,wherein said T cells are unfractionated T cells, isolated CD8+ T cells,or isolated CD4+ T cells.

151. The centrifugal chamber of any of embodiments 147-150, wherein atleast 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, or atleast 75% of said cells in said composition are transduced with a viralvector.

152. The centrifugal chamber of any of embodiments 147-151, wherein:

the viral vector encodes a recombinant receptor; and

cells in the composition express the recombinant receptor.

153. The centrifugal chamber of embodiment 151, wherein said recombinantreceptor is a recombinant antigen receptor.

154. The centrifugal chamber of embodiment 153, wherein said recombinantantigen receptor is a functional non-T cell receptor.

155. The centrifugal chamber of embodiment 154, wherein said functionalnon-T cell receptor is a chimeric antigen receptor (CAR).

156. The centrifugal chamber of any of embodiments 151-155, wherein saidrecombinant receptor is a chimeric receptor comprising an extracellularportion that specifically binds to a ligand and an intracellularsignaling portion containing an activating domain and a costimulatorydomain.

157. The centrifugal chamber of embodiment 153, wherein said recombinantantigen receptor is a transgenic T cell receptor (TCR).

158. The centrifugal chamber of any of embodiments 147-157, wherein:

among all the cells in the composition, the average copy number of saidrecombinant viral vector is no more than about 10, no more than 8, nomore than 6, no more than 4, or no more than about 2; or

among the cells in the composition transduced with the recombinant viralvector, the average copy number of said vector is no more than about 10,no more than 8, no more than 6, no more than 4, or no more than about 2.

159. A centrifugal chamber rotatable around an axis of rotation, saidchamber comprising an internal cavity comprising the composition of anyof embodiments 133-145.

160. The centrifugal chamber of embodiment 159, further comprising avolume of gas up to the maximum volume of the internal cavity of thechamber.

161. The centrifugal chamber of embodiment 160, wherein said gas is air.

162. The centrifugal chamber of any of embodiments 146-161, said chamberbeing rotatable around an axis of rotation and comprising an end wall, asubstantially rigid side wall extending from said end wall, and at leastone opening, wherein at least a portion of said side wall surrounds saidinternal cavity and said at least one opening is capable of permittingintake of liquid into said internal cavity and expression of liquid fromsaid cavity.

163. The centrifugal chamber of any of embodiments 146-162, wherein saidside wall is curvilinear.

164. The centrifugal chamber of embodiment 163, wherein said side wallis generally cylindrical.

165. The centrifugal chamber of any of embodiments 162-164, wherein

said at least one opening comprises an inlet and an outlet, respectivelycapable of permitting said intake and expression; or

said at least one opening comprises a single inlet/outlet, capable ofpermitting said intake and said expression.

166. The centrifugal chamber of any of embodiments 162-165, wherein saidat least one opening is coaxial with the chamber and is located in theend wall.

167. The centrifugal chamber of any of embodiments 162-166, wherein saidcentrifugal chamber further comprises a movable member and said internalcavity is a cavity of variable volume defined by said end wall, saidsubstantially rigid side wall, and said movable member, said movablemember being capable of moving within the chamber to vary the internalvolume of the cavity.

168. The centrifugal chamber of embodiment 167, wherein:

the movable member is a piston; and/or

the movable member is capable of axially moving within the chamber tovary the internal volume of the cavity.

169. The centrifugal chamber of any of embodiments 162-168, wherein:

the internal surface area of said cavity is at least at or about 1×10⁹μm²;

the internal surface area of said cavity is at least at or about 1×10¹⁰μm²;

the length of said rigid wall in the direction extending from said endwall is at least about 5 cm;

the length of said rigid wall in the direction extending from said endwall is at least about 8 cm; and/or

the cavity comprises a radius of at least about 2 cm at least onecross-section.

170. The centrifugal chamber of any of embodiments 159-169, wherein theliquid volume of said composition present in said cavity is between orbetween about 0.5 mL per square inch of the internal surface area of thecavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in.

171. The centrifugal chamber of any of embodiments 159-169, wherein theliquid volume of said composition present in said cavity is at least 0.5mL/sq.in., 1 mL/sq.in., 2.5 mL/sq.in., or 5 mL/sq.in.

172. A closed system, comprising the centrifugal chamber of any ofembodiments 147-158 and 162-171.

173. The closed system of embodiment 172, further comprising a multi-waymanifold operably connected to one or a plurality of containers.

174. The closed system, comprising the centrifugal chamber of any ofembodiments 159-171.

175. The closed system of embodiment 174, further comprising a sterilefilter.

176. The closed system of any of embodiments 172-175, wherein thecentrifugal chamber is capable of rotation at a speed up to 8000 g,wherein the centrifugal chamber is capable of withstanding a force of upto 500 g, 600 g, 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 2000 g,2500 g, 3000 g or 3200 g, without substantially yielding, bending, orbreaking or otherwise resulting in damage of the chamber and/or whilesubstantially holding a generally cylindrical shape under such force.

177. The method of embodiment 49, wherein at least at or about 30, 40,50, 60, 70, 80, or 80% of the T cells in the output composition comprisehigh expression of CD69 and/or TGF-beta-II.

178. The method of embodiment 177, wherein said at least 30, 40, 50, 60,70, 80, or 80% of the T cells in the composition comprise no surfaceexpression of CD62L and/or comprise high expression of CD25, ICAM,GM-CSF, IL-8 and/or IL-2.

179. A method comprising

washing primary human cells; and

incubating said cells with a selection reagent under agitationconditions whereby at least a plurality of the human cells arespecifically bound by the selection reagents,

wherein said washing and incubating are carried out within a closed,sterile system and at least in part in an internal cavity of acentrifugal chamber integral to the closed, sterile system.

180. The method of embodiment 179, wherein the method steps are carriedout in an automated fashion based on input from a user that the methodshould be initiated, resulting in completion of the method steps.

181. The method of embodiment 105, 179 or 180, wherein the incubationunder mixing conditions comprises effecting rotation of the chamber forat least a portion thereof.

182. The method of embodiment 181, wherein the effecting rotation for atleast a portion thereof comprises effecting rotation at a plurality ofperiods during the incubation, said plurality of periods being separatedby one or more periods of rest, at which the chamber is not rotated.

183. The method of embodiment 182, wherein one or more or all of theplurality of periods of effecting rotation is for a time that is or isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as 1 or 2 secondsand/or one or more or all of the one or more periods of rest is for atime that is or is about 3, 4, 5, 6, 7, 8, 9, or 10 or 15 seconds, suchas 4, 5, 6, or 7 seconds.

184 The method of any of embodiments 105 or 179-183, wherein theincubation under mixing conditions is carried out for at least orapproximately 10, 15, 20, 30, or 45 minutes, such as at or about 30minutes.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Viral Transduction of Primary Human T Cells in a CentrifugalChamber

This example demonstrates transduction of isolated primary human T cellswith a recombinant viral vector encoding a chimeric antigen receptor(CAR), with transduction initiated under centrifugal force in asubstantially rigid cylindrical centrifugal chamber, according to anembodiment provided herein. T cells were isolated via positive selectionfrom a human apheresis product sample.

The resulting cells were activated using an anti-CD3/CD28 reagent. Forinitiation of transduction, the cells were incubated with a viralparticle containing a viral vector genome encoding an anti-CD19 CARunder various conditions following activation.

Under one set of conditions (“Sepax”), transduction was initiated byincubating the cells in a cavity of a centrifuge chamber (Biosafe SA,A200), under centrifugation in a Sepax® 2 processing unit (Biosafe SA).A 50 mL liquid composition containing 50×10⁶ of the isolated cells wascombined in a 300 mL transfer pack with 50 mL liquid stock containingthe viral vector particles. Using the Sepax® system to move the pistonof the chamber, the composition was pulled into the cavity of thecentrifuge chamber. 100 mL air also was pulled in, thereby increasingthe volume of the cavity to 200 mL and resulting in a decrease in theratio of the volume of liquid in the cavity to the internal surface areaof the cavity. The chamber was spun by ramping up to a speed of atapproximately 4600 rpm on the Sepax® 2 unit, corresponding to a relativeg force (relative centrifugal force (RCF)) at the internal side wall ofthe processing cavity of the chamber of approximately 600. The durationof the spin at this speed was 60 minutes.

For another set of conditions (“VueLife”), a composition containing25×10⁶ cells and the same stock of viral vector particles at a 1:1volumetric ratio were incubated in 50 mL in a centrifuge bag, in a CI-50centrifuge adapter, and spun at an approximate relative centrifugalforce (RCF) on the cells of approximately 1000 g for 60 minutes. A bagwith a smaller volume compared to the centrifuge chamber was used inorder to permit centrifugation at a relative centrifugal force on thecell of 1000 g. Controls included an “untransduced” sample (same cellconcentration incubated for the same time in a 24-well plate withoutvirus without centrifugation and a “no-spin” control (“0xg”) sample(same cell/virus concentration incubated for the same time in the sameplate without centrifugation). Under each set of conditions, apolycation was included. Following spin (or comparable “no-spin”incubation), the compositions were incubated for 24 hours at 37 degreesC. to complete transduction.

The cells were expanded and transduction efficiency for each of therespective conditions was calculated on Day 6 post-isolation aspercentage of CD3⁺ T Cells with surface expression of the encoded CAR(as detected by flow cytometry using an antibody specific for the CAR).The results are shown in FIG. 1A. As shown, greater transductionefficiency was observed following the initiation of transduction byincubating cells in the cavity of the centrifuge chamber under rotation,as compared to in the centrifuge bag (VueLife®) and controls. FIG. 1Bshows cell expansion (as indicated by number of population doublings)over the six-day period.

Example 2: Transduction of Primary Human T Cells in a CentrifugalChamber at Different Ratios of Liquid Volume to Surface Area

Transduction efficiency following initiation of transduction in thecentrifuge chamber was assessed under various conditions, using the samenumber of cells and infectious units of virus, and different ratios ofliquid volume to internal surface area of the chamber's cavity. Cellswere generally prepared and stimulated as described in Example 1. Alltransduction initiation conditions used an IU:cell ratio of 2:1 and atotal number of 100×10⁶ cells.

For a first sample (“5.1 mL/sq.in.,” referring to the 5.1:1 mL of liquidper square inch of internal cavity surface used in this condition),100×10⁶ cells, in a liquid volume of 100 mL, were combined with 100 mLof a liquid composition containing the viral vector particles. For asecond sample (“2.5 mL/sq.in.,” referring to 2.5 mL of liquid per squareinch of cavity surface used in this condition), 50 mL of a liquidcomposition with the same number of cells was combined with 50 mL of aliquid composition containing the viral vector particles. In each case,a polycation was included for a final concentration duringcentrifugation of 10 μg/mL. The respective liquid compositions (and forthe second sample, 100 mL of air) were drawn into and incubated in theliquid-holding cavity of the chamber. In each case, the chamber was spun(by ramping up) in the Sepax® 2 processing unit at an rpm ofapproximately 4600, corresponding to an RCF at the internal cavity sidewall of approximately 600 g for 60 minutes. The samples then wereincubated for an additional 24 hours at 37 degrees, for completion oftransduction.

The cells were expanded and transduction efficiency calculated on Day 6post-isolation, by determining the percentage of CD3⁺ T Cells withsurface expression of the CAR, detected as described above. The resultsare shown in FIG. 2. As shown, for initiation of transduction in thecentrifugal chamber using the same number of cells and infectious unitsof virus, a greater transduction efficiency was observed when using alower ratio of liquid volume of the composition in the cavity to theinternal surface area of the cavity.

Example 3: Transduction of Primary Human T Cells in a CentrifugalChamber

Another study compared transduction efficiency under various conditions,including transduction in a centrifugal chamber according to embodimentsof the provided methods, using various ratios of liquid volume to cavitysurface area. Human T cells were isolated from an apheresis product andstimulated as described above.

Following the stimulation, 80×10⁶ cells were incubated under varyingconditions, including for transduction with a viral vector encoding aCAR. A polycation was included in all samples.

For conditions under which transduction was initiated in the centrifugechamber, 80×10⁶ cells were incubated with virus containing the vector inthe cavity of the chamber, at a ratio of 2 IU virus per cell. Theincubation was carried out while centrifuging the chamber using theSepax® 2 Processing system at an RCF at the internal side wall of thecavity of approximately 600 g for 60 minutes. Under one set ofconditions (“Sepax (0.1 IU/cell/mL),” with 0.5 mL liquid volume persquare inch of internal cavity surface), for centrifugation, the cellsand virus were pulled into the cavity of the chamber in a total liquidvolume of 20 mL; 180 mL of air also was pulled into the cavity. Underanother set of conditions (“Sepax (0.01 IU/cell/mL),” with 5.1 mL liquidvolume per square inch of internal cavity surface), the same number ofcells and infectious units of virus were pulled in in a 200 mL liquidvolume.

Under separate conditions, “1000 g in plate,” transduction of cells wasinitiated in the presence of virus (2 IU/cell) in a 24-well plate, withcentrifugation at an RCF on the cells of approximately 1000 g for 60minutes. An “untransduced” negative control (incubation in a 24-wellplate without virus or centrifugation) and a “no spin” control(incubation with virus at a ratio of 2 infectious units (IU) per cellwithout centrifugation in the same 24-well plate) also were used. Cellswere incubated for 24 hours at 37 degrees C. to complete transduction.

Cells were expanded and transduction efficiency for each samplecalculated as percent of CD3⁺ cells expressing the CAR on their surface,as described in Examples 1 and 2. The results are shown in FIG. 3. Asshown, transduction was observed following initiation of transductionunder rotation in the centrifuge chamber and in the 24-well plate ascompared to the control conditions. For transduction initiation in thecentrifuge chamber, greater transduction efficiency was observed with alower ratio of liquid volume to internal surface area of the chambercavity.

Example 4: Assessment of Vector Copy Number (VCN) Following Transductionin a Centrifugal Chamber

Copy number of the integrated viral vector (VCN) was assessed followingtransduction initiated under certain conditions in the study describedin Example 3. VCN per cell was determined for the SGF-derived retroviralvector by real-time quantitative PCR (RT-qPCR). Mean VCN was determinedby qPCR specific for viral genome among all cells in the compositionfollowing transduction (“VCN/cell”), and separately among the transducedcells (cells expressing the transgene) alone (“VCN/CAR”). The resultsare presented in FIG. 4. In the graph shown in FIG. 4, the label “Sepax20” refers to a 20 mL liquid volume used in the chamber cavity duringtransduction initiation; the results so-labeled are from the same studyand condition labeled as “Sepax (0.1 IU/cell/mL)” in Example 3 (forwhich transduction efficiency was determined to be 25%). Similarly, thelabel “Sepax 200” refers to a 200 mL liquid volume used in the chambercavity during transduction initiation; the results so-labeled are fromthe same study and conditions labeled as “Sepax (0.01 IU/cell/mL)” inExample 3 (for which the transduction efficiency was determined to be7%). As shown, when transduction was initiated by initiatingtransduction of cells in the cylindrical, substantially rigid centrifugechamber under rotation, increased transduction efficiency was notassociated with increased vector copy number. In this study, theconditions producing increased transduction efficiency also produceddecreased mean vector copy number per cell.

Example 5: Transduction Using a Sepax® 2 Processing System

In an exemplary process, T cells are transduced with a viral vectorparticle in an automated fashion in a centrifugal chamber integral to asingle-use system and the Sepax® 2 processing system (Biosafe SA). Thechamber is integral to a sterile, disposable closed system, which is asingle-use processing kit sold by Biosafe SA for use in regenerativemedicine. The kit is configured to include a series of tubing linesconnecting the chamber to a series of containers, with a generalconfiguration shown in FIG. 5 and/or FIG. 7. The chamber (1) includes anend wall (13) including an inlet/outlet opening (6), a rigid side wall(14), and a piston (2), which collectively define an internal cavity (7)of the chamber. The system is configured to include various containerslabeled: Output Bag, Waste Bag, Input Bag, and two diluent bags (DiluentBag 1 and Diluent Bag 2), and various connectors, including stopcocks,and valves. Clamps (5) are included for blocking flow between differentportions of the system via the tubing lines. In some embodiments, thesystem includes a male luer lock sterile filter (15) with female luerlock cap (16), through which gas, e.g., air, may be drawn in a sterilemanner, when the cap is released/removed. The system is placed inassociation with the Sepax® 2 processing unit, including a centrifugeand cabinet for housing components.

In the exemplary process, the Input Bag contains a compositioncontaining the cells to be transduced. Diluent Bag 1 contains viralvector particles, polycation, and medium. In some embodiments, air isincluded in the bag with the vector particles. For example, in analternative embodiment, a container with air and/or additional mediummay be connected at this position instead of and/or in addition to thevirus composition.

A user indicates to the processing unit via a user interface that a newprogram is to be run and inputs various parameters into the system,including an Initial Volume (between 20-900 mL), a Final Volume (between20-220 mL), an Intermediate Volume (between 10-100 mL), a DilutionVolume (between 50-220 mL), a g-force (between 100-1600 or between200-3200 g) (RCF at the internal side wall of the processing cavity ofthe chamber)), and a Sedimentation Time (between 120 and 3600 seconds).The user indicates to the system that the process should be initiated,inputs identification information for the subject from which cells arederived, and indicates to the system that input is complete, whichprompts the system to carry out a test of the closed system kit.

With all respective stopcock valves in the closed position, clampsblocking movement of fluid between the tubing lines and Diluent Bag 1,Waste Bag, Input Bag, and Output Bag (for collection of the productcontaining the cells), respectively, are opened and an automated programinitiated by communication with the system by the user.

In response, the system causes, in an automated fashion, movement ofliquid and/or gas between the various components of the closed system bycausing opening and closing of the valves and movement of the piston tovary the volume of the cavity. It causes repositioning of a stopcock topermit flow between the Input Bag and the internal cavity of thechamber, via the inlet/outlet opening and lowering of the piston withinthe centrifugal chamber, thereby increasing the volume of the cavity anddrawing a volume (the user-defined Initial Volume) of the composition ofcells and viral vector particles from the Input bag to the processingcavity, via an inlet/outlet in the end wall of the chamber.

The system prompts the centrifuge to spin the chamber for 120 seconds at500 g, prompts purging of 20 mL volume from the cavity into the InputBag to rinse it, and drawing of the volume back into the cavity. Thesystem prompts the centrifuge to spin the chamber for 180 seconds at 500g, causing sedimentation. The system repositions the stopcocks to permitflow of fluid and/or gas between the cavity and the Waste Bag andeffects extraction of fluid from the cavity into the Waste Bag, leavingthe user-defined Intermediate Volume in the cavity.

The system causes rotation of the stopcock to block movement of fluidbetween the tubing and the waste bag. The system causes intake of viralvector particle-containing liquid composition and, if applicable, air(collectively, at the user-defined Diluent Volume) from Diluent Bag 1 tothe cavity of the chamber. These steps collectively effect intake of aninput composition containing cells to be transduced and viral vectorparticles and in some cases, air, into the cavity. In some embodiments,the total volume of the cavity is 200 mL, for example, including 200 mLliquid volume or including less than 200 mL liquid volume and theremainder of the cavity volume including air.

Centrifugation of the chamber is carried out for the user-definedSedimentation Time at the user-defined g-force, resulting in initiationof transduction of cells in the input composition with viral vectorparticles. In an alternative embodiment, a volume of air and/or mediumis pulled into the cavity from another bag at the position of DiluentBag 1 and 2, prior to centrifugation. In an alternative embodiment, airis drawn in prior to centrifugation through the luer lock filter (15),e.g., by the user opening the clamp (5) blocking movement of fluidbetween the filter (15) and tubing lines and the cavity (7) andreleasing the female cap (16), allowing air remaining in the chamber topass in through the filter from the environment. In some embodiments,the movement of air is automated by the system, for example, based on anadditional user-defined air input volume inputted into the system andthe user indicating to the system that air may be taken in at thedefined air volume. In some embodiments, air, if present, is releasedthrough the tubing line and uncapped filter (15) by a similar processfollowing centrifugation.

When prompted by the system, the user closes the clamp blocking movementof fluid between Diluent Bag 1 and the tubing lines and opens the clampblocking movement of fluid between Diluent Bag 2 and the tubing lines.The system causes movement of fluid from Diluent Bag 2 to the processingcavity by opening of the appropriate stopcock valve and movement of thepiston to draw into the cavity a volume of fluid from Diluent Bag 2equal to the amount required to result in a total liquid volume in thechamber equal to the user-defined Final Volume. The system then causesmixture of the fluid in the cavity for 60 seconds and then transfer ofthe fluid in the internal cavity to the Output Bag, which therebycontains an output composition with cells to which viral particles havebound and/or infected with the viral vector. These cells then aregenerally incubated for completion of transduction, for example, at 37degrees C., for example, for 24 hours.

Example 6: Assessment of Cell Growth and Viability at DifferentCentrifugal Forces

The effect of centrifugal force on cells during the centrifugation usedto initiate transduction of cells in a chamber according to certainprovided embodiments was assessed. Cell expansion and cell viabilitywere assessed upon exposure to different centrifugal forces.

T cells were isolated and stimulated essentially as described inExample 1. At day 4, various, each individually containing the cells,were pulled into a cavity of a centrifuge chamber in a Sepax® 2processing unit (Biosafe SA) and subjected to centrifugation at variouscentrifugal forces. Specifically, samples were spun for 60 minutes in achamber (A-200F) integral to a single-use kit using the Sepax® 2processing system at approximately 4600 rpm, approximately 6000 rpm, andapproximately 7400 rpm, respectively), which achieved an RCF at theinternal surface of the side wall of the cavity of approximately 600 g1000 g, and 1600 g, respectively. As a control, a sample of the cellswas separately pulled into the centrifuge cavity, but not spun (0 gcondition). In each case, after the spin (or incubation with no spin),the cells were incubated at 37° C., 5% CO₂, through day 10. At variouspoints throughout the process, cell expansion (population doublings ascompared with cell number at day 0) and viability were monitored.Specifically, these measurements were taken at days 0, 3, 4, 5, 6, 7,and 10. The results are shown in FIG. 6.

As shown in FIG. 6A and FIG. 6B, respectively, centrifugation at thevarious speeds was observed to have no substantial effect on cellexpansion (“population doublings”) or viability over the 10 days. Theresults demonstrate that the T cells could tolerate centrifugation atrelative centrifugal forces of at least up to or about 1600 g, asmeasured at the side wall of the cavity of the chamber, corresponding toapproximately the same average force on the cells at the cellsurface:liquid interface, under conditions used for transductioninitiation in embodiments provided herein, without detectablesubstantial changes in expansion or viability.

Example 7: Transduction Process Step Using Transduction Initiation inGenerally Cylindrical Centrifugal Chamber

This example describes the general parameters of a transduction processstep that was used in the studies described in Examples 8-10.Transduction of cells with a recombinant viral vector encoding achimeric antigen receptor (CAR) was initiated in a centrifugal chamberaccording to provided embodiments.

CD4⁺/CD8⁺ T cells were isolated via positive selection from a humanapheresis product sample. The isolated cells were cryopreserved andthawed at 37° C. The thawed cells were activated using CD3/CD28 beads inthe presence IL-2 (100 IU/mL) for 72 hours at 37° C. prior to initiationof transduction. In some cases, various aspects of the apheresispreparation, isolation, and/or activation steps also were carried out inthe cavity of a centrifugal chamber according to provided embodiments,in association with the Sepax® 2 system, e.g., as described in Example11.

In preparation for transduction, a centrifugal processing chamber (1)(A-200F), integral to a sterile, single-use disposable kit sold byBiosafe SA for regenerative medicine use, essentially as depicted inFIG. 7, was placed in association with a Sepax® 2 processing unit, whichthus could provide to the chamber centrifugal force and axialdisplacement (permitting control of the dimensions of the internalcavity). (U.S. Pat. No. 6,733,433).

To initiate transduction, the following steps were carried out.

To generate a composition containing viral vector particles for sterilemixing with the activated cells in the centrifuge chamber, completemedia (containing serum free hematopoietic cell medium supplemented with5% human serum, and IL-2, and a polycation in an amount sufficient for afinal concentration during transduction initiation of 10 μg/mL), viralvector particles at the indicated number or relative number ofinfectious units (IU) (for example, 1.8 IU/cell or 3.6 IU/cell), and,where applicable, air, were aseptically transferred to a centrifuge bag,which ultimately would be sterilely connected with the kit at a DiluentBag position for intake as described below.

A culture bag containing the activated cells was sterilely connected tothe single-use disposable kit via tubing line at the position of the“Input Bag” shown in FIG. 5 and FIG. 7. An automated “dilution” protocolwas run on the Sepax® 2 processing unit. Thereby, through movement ofthe piston, the desired number of cells (as indicated in individualstudies described, for example, 50×10⁶, 100×10⁶ or 200×10⁶ cells) wastransferred from the culture bag to a product bag at the “Output Bag”position shown in FIG. 5 and FIG. 7, by way of the chamber cavity.

To generate an input composition with both cells and virus (and whereapplicable, air) for intake into the chamber and transduction, theproduct bag containing the desired number of activated cells then wassterilely connected at the Input Bag position as shown in FIG. 5 andFIG. 7. The centrifuge bag containing the viral particles, media, andoptionally air was sterilely connected at the position of Diluent Bag 1shown in the figures. An automated Wash cycle was run on the Sepax® 2,facilitating drawing in of the composition containing the cells into thecavity of the chamber, spinning of the composition on the Sepax® 2 at anapproximate RCF at the internal wall of the cavity of 500 g to pelletthe cells, and removal of the appropriate volume of liquid required toachieve a desired volume, e.g., 10 mL. The contents of the centrifugebag at the Diluent Bag 1 position, including the viral particles, media,polycation, and where applicable, air, then was drawn into the cavity ofthe chamber with the cells. This process thus effected avolume-reduction of the cell composition and combined the volume-reducedcells with the virus-containing composition and, where applicable, air.The resulting 200 mL volume (containing the cells, virus, andoptionally, air) then was transferred into a centrifuge bag in the“Output Bag” position of the kit as shown in FIG. 5 and FIG. 7.

To initiate transduction, the centrifuge bag containing 200 mL of thevirus, cells, and air where applicable then was removed and sterilelyconnected at the Input Bag position of the kit. A bag containingcomplete media was sterilely connected to the kit at a “Diluent Bag”position. A cell culture bag was sterilely connected to the system atthe “Output Bag” position. The user indicated via the interface that anautomated protocol should be run on the system for initiation oftransduction. Specifically, the program caused transfer of the 200 mLvolume containing cells, virus, and where indicated, air, via the tubinglines to the cavity of the chamber by movement of the piston. Theprogram continued with centrifugation of the contents in the cavity ofthe chamber (total volume 200 mL) at the indicated force, to initiatetransduction of cells with the viral vector particles. In someembodiments, a hand-held laser tachometer was used to verify revolutionsper minute (rpm) at various set points on the Sepax® unit using knownmethods. Except where specifically indicated, the spin was carried outat the indicated speed for 1 hour (3600 s), with additional ramp-up andramp-down time. Following the spin and when prompted by the system, theuser closed the clamp permitting movement of fluid between the cavityand the Input Bag and opened the clamp blocking movement of fluidbetween the cavity and product bag at the Output Bag position. Uponinput from the user, the program continued by effecting movement ofliquid from the chamber to the output bag.

Where applicable, for expulsion of air, when prompted by the system, theuser opened the clamp blocking movement of fluid between the chambercavity and the filter, and the program caused expression of air via thefilter.

A dilution program then was run on the Sepax® 2 system, with the clampblocking movement of fluid between the diluent bag with the media andthe chamber opened and the program causing movement (by opening ofstopcock(s) and movement of the piston) of the appropriate amount ofliquid from that bag to the chamber, mixing for 60 seconds, and thentransfer of the fluid from the processing cavity to the output bag, theappropriate amount being that needed to achieve a user-defined FinalVolume of 200 mL, given the presence of air during centrifugation, ifany.

The culture bag in the Output Bag position thereby contained an outputcomposition with cells containing bound viral particles and/orinoculated with the viral genome. The cells then were incubated in thebag for ˜24 hours at 37 degrees C., 5% CO₂, for completion oftransduction. During the transduction initiation and completion, viralvector particles inoculated cells and their genomes became integratedinto the cellular genomes, as indicated by the various measures fortransduction efficiency and copy number in the individual examples.

Example 8: Transduction Initiation in a Centrifugal Chamber withConstant Volume and Viral Particle Number and Different CellConcentrations

Compositions with various cell numbers and infectious units (IU) ofviral particles, in constant liquid volume, were subjected to thetransduction process described in Example 7. In each case, prior totransduction initiation, cells were collected, washed, isolated,cryopreserved, and activated as described in Example 11.

Example 8A

Transduction initiation and further culture to complete transductionwere carried out as described in Example 7, with the followingspecifics.

Under two separate conditions in two separate studies, the transductioninitiation process was carried out in a 70 mL total liquid volume (theremaining 130 mL volume of the cavity during spin containing air). Theseparate conditions were carried out on 200×10⁶ cells and 100×10⁶ cells,respectively. The same total number of units of viral vector particlescontaining vectors encoding anti-CD19 CAR were used, resulting in 1.8IU/cell and 3.6 IU/cell for the two conditions, respectively. During thetransduction program, the 3600 second spin was at approximately 7400rpm, corresponding to an RCF of approximately 1600 g at the side wall ofthe processing cavity.

After the ˜24-hour incubation, the cells were expanded in a BioreactorSystem with perfusion. Transduction efficiency for the respectivecompositions was calculated on Day 6 as percentage of CD3⁺ T Cells withsurface expression of the encoded CAR, detected as described in Example1, and was compared to an untransduced population of cells as a control(“untransduced”). The results are shown in FIG. 8A. As shown, with thesame total volume and total number of infectious units during incubationunder rotation, a greater transduction efficiency was observed for thecondition using a smaller number of 100×10⁶ cells in the cavity duringthe incubation.

Example 8B

In another study, transduction initiation and further culture tocomplete transduction were carried out as described in Example 7, withthe following specifics.

Under three separate conditions, the transduction initiation process wascarried out in a 70 mL total liquid volume (the remaining 130 mL volumeof the cavity during spin containing air). The separate conditions werecarried out on 200×10⁶ cells, 100×10⁶ cells, and 50×10⁶ cells,respectively. The same total number of units of viral vector particlescontaining vectors encoding anti-CD19 CAR were used, which was thenumber of units needed to result in 1.8 IU/cell for the condition with200×10⁶ cells. During the transduction program, the 3600 second spin wascarried out on the Sepax® 2 system at approximately 7400 rpm,corresponding to an RCF of approximately 1600 g at the side wall of theprocessing cavity.

After the ˜24-hour incubation, the cells were expanded in a BioreactorSystem with perfusion. Transduction efficiency for the respectivecompositions was calculated on Day 6 as percentage of CD3⁺ T Cells withsurface expression of the encoded CAR, detected as described in Example1, and was compared to an untransduced population of cells as a control(“untransduced”). The results are shown in FIG. 8B. As shown, with thesame total volume and total number of infectious units during incubationunder rotation, a greater transduction efficiency was observed for thecondition using a smaller number of 100×10⁶ cells in the cavity duringthe incubation.

Vector copy number (VCN) also was assessed in transduced cells at day 6,as described in Example 4, with mean VCN determined among the transducedcells (cells containing SGF+ viral vector nucleic acid in their genome).An untransduced cell control (“PD UnTD”) and positive control cells (“2Copy Positive Control”) also were assessed. The results are presented inFIG. 8C.

Example 9: Transduction Initiation in Centrifugal Chamber with VariousVolumes and Units of Viral Vector Particles

Compositions with increasing numbers of infectious units (IU) of viralparticles and liquid volumes (with constant number (100×10⁶) of cells)were subjected to the transduction process described in Example 7, withthe following specifics. In each case, prior to transduction initiation,cells were collected, washed, isolated, cryopreserved, and activated asdescribed in Example 11.

In the process described in Example 7, the composition containing theviral particles, media, and air that was drawn in from the Diluent Bagposition for combining with the cells via the dilution protocol,included 60, 90, and 120 mL liquid volumes, respectively, for thedifferent conditions (with the 10 mL cell-containing composition,resulting in 70, 100, and 130 mL liquid volume, respectively, for theindividual conditions), with the remaining of the 200 mL total volumepulled into the chamber for spinning being comprised of air. Each ofthese liquid volumes included 6×10⁶ IU viral vector particles per mL ofliquid volume, resulting in an increasing IU and IU/cell for eachcondition. The speed for the 3600 second spin was carried out atapproximately 7400 rpm, corresponding to an RCF of approximately 1600 gat the internal wall of the cavity on the Sepax® unit. An untransduced(“mock”) control also was used.

After the ˜24-hour incubation, the cells were expanded in a BioreactorSystem with perfusion. Transduction efficiency for the respectivecompositions was calculated on Day 6 as percentage of CD3⁺ T Cells withsurface expression of the encoded CAR, detected as described inExample 1. The results are shown in FIG. 9A. As shown, for the samenumber of cells, an increasing amount of virus with correspondingincrease in volume resulted in an increased transduction efficiency inthis study.

Mean vector copy number (VCN) per transduced cell (cells expressing thetransgene) also was determined at day 6 for each condition by real-timequantitative PCR (RT-qPCR) as described in Example 4. The results arepresented in FIG. 9B.

Example 10: Effect of Centrifugation Time on Transduction EfficiencyUsing a Centrifugal Chamber

100×10⁶ cells were subjected to transduction as described in Example 7,with various durations used for the incubation under centrifugation.Specifically, the total liquid volume used for the centrifugation in theprocessing cavity of the chamber was 70 mL (with the remaining 130 mL ofthe 200 mL total volume composed of air). Viral vector particlescontaining a vector encoding an anti-CD19 CAR were included in thisvolume at a ratio of 3.6 IU/cell. In each case, prior to transductioninitiation, cells were collected, washed, isolated, cryopreserved, andactivated as described in Example 11.

The spin for initiation of transduction was carried out at approximately7400 rpm, corresponding to an approximately a 1600 g relativecentrifugal force on the inner side wall of the processing chamber. Theduration of the spin at this speed was 10 minutes for one condition and60 minutes for the other.

After the ˜24-hour incubation, the cells were expanded in a BioreactorSystem with perfusion. Transduction efficiency for the respectivecompositions was calculated on Day 6 as percentage of CD3⁺ T Cells withsurface expression of the encoded CAR detected as described inExample 1. An untransduced control also was assessed (“untransduced”).The results are shown in FIG. 10. As shown, in this study, greatertransduction efficiency was observed following initiation oftransduction of 100×10⁶ cells in the processing cavity of the centrifugechamber under centrifugation for 60 minutes as compared to 10 minutes.

Example 11: Preparation of Genetically Engineered Cells

This example describes an exemplary process which has been carried outto prepare, from a biological sample, genetically engineered T cellstransduced with a nucleic acid encoded by a viral vector, according tocertain embodiments provided herein. As described in individualexamples, prior to the transduction steps carried out in studiesdescribed in various examples herein, some of the steps of this processwere carried out, for example, collection, wash, cryopreservation,selection, and activation steps, as described in this example.

Various steps of the process were carried out within the processingcavity of a centrifugal chamber having a rigid, generally cylindricalside wall and a piston capable of moving within the chamber to vary thevolume of the cavity (the processing cavity of a Sepax® centrifugechamber contained within a single-use kit). Specifically, steps carriedout in the chamber included cell washing, dilution/buffer-exchange,steps for affinity-based selection (e.g., incubation with immunospecificbinding agents), transduction initiation, formulation, and steps foractivation/expansion (e.g., incubation with stimulatory agent(s)).

1. Sample Collection and Leukapheresis

A human leukapheresis sample enriched in mononuclear cells was obtainedfrom a whole blood sample from a subject using a leukapheresiscollection system. The leukapheresis sample was stored sealed at 2-8°C., for no more than about 48 hours.

2. Leukapheresis Wash

The leukapheresis sample was sterilely transferred to a transfer pack.Cells of the leukapheresis sample were washed and resuspended in abuffer for use in affinity-based selection, the buffer containing PBS,EDTA, and human serum albumin. The wash was carried out within asterile, single-use disposable kit sold by Biosafe SA for use inregenerative medicine, which included a centrifugal chamber (1),essentially as depicted in FIG. 7. The transfer pack containing thecells and a bag containing the buffer were sterilely connected to thekit, which was placed in association with a Sepax® 2 processing unit.The wash and resuspension were carried out using a standard cell washprotocol on the unit, with the cells retained in the processing cavity(7) of the centrifuge chamber at the end of the protocol, for subsequentincubation with reagents for affinity-based selection (see 3).

3. Affinity-Based Selection

For positive, immunoaffinity-based selection of T cells, the sameautomated program was continued to incubate the washed cells in theselection buffer with magnetic beads coupled to monoclonal antibodiesspecific for CD4 and CD8. The incubation was carried out at roomtemperature in the same centrifugal chamber (1) in which the cells wereretained after the wash (see 2) described above. Specifically, the beadswere mixed in selection buffer in a transfer pack, which then wassterilely connected at a Diluent Bag position of the single-use kit usedfor the wash step. A program was run on the Sepax® 2 unit which causedthe bead mixture and selection buffer to be drawn into the chamber withthe washed cells, and the contents of the chamber (total liquid volume100 mL) to be mixed for 30 minutes, via a semi-continuous process. Themixing was carried out with repeated intervals, each including shortduration (approximately 1 second) centrifugation at low speed(approximately 1700 rpm), followed by a short rest period (approximately6 seconds).

At the end of the program, the Sepax® 2 unit caused pelleting of thecells and expulsion of excess buffer/beads into a bag at the Waste Bagposition, washing of the pelleted cells, and resuspension in selectionbuffer. The wash was carried out on the Sepax® at an RCF at the internalwall of the cavity of approximately 200 g, for 180 seconds. The programcaused the washed cells to be collected into a transfer pack placed atthe Output Bag position in the exemplary kit shown in FIG. 7, thecontents of which could be transferred via tubing lines to a column formagnetic separation, within a closed system. Thus, the cell wash andincubation with the affinity-based selection reagent was carried outentirely within the same closed, sterile system, by passing liquid andcells to and from the cavity of the centrifugal chamber. The ability tocontrol and adjust liquid volumes and to mix the cells under rotation inthe chamber allowed use of substantially less of the selection reagentper cell processed as compared to incubation in a tube with shaking orrotation.

The cells then were passed from the transfer pack, through a closed,sterile system of tubing lines and a separation column, in the presenceof a magnetic field using standard methods, to separate cells that hadbound to the CD4- and/or CD8-specific reagents. Thesemagnetically-labeled cells then were collected in a transfer pack forfurther processing.

4. Cryopreservation

The transfer pack with the labeled, selected cells wassterilely-connected to a single-use disposable kit sold by Biosafe ASfor regenerative medicine for use with the Sepax® 2 system. The kit wasessentially as shown in FIG. 7, except that two ports, as opposed toone, were present at the position to which the Output Bag is attached inthe exemplary system shown in FIG. 7, with a collection bag sterilelyconnected at each port; two ports, as opposed to one, were present atthe position to which the Input Bag is connected in FIG. 7; and a singleport, as opposed to two, were present at the position of Diluent Bags 1and 2 in FIG. 7. A standard wash cycle was carried out on the Sepax® 2unit to reduce the volume of the washed cells. A bag with cryomedia wassterilely connected to the kit and a dilution protocol run twice totransfer the cryomedia to the cell composition and expel the resultingcomposition into the two output cryopreservation bags. The cells in thecryopreservation bags were cryopreserved and stored in liquid nitrogenuntil further use.

5. Thaw and Activation

Cryopreserved cells for were thawed. The thawed cells were activatedusing an anti-CD3/CD28 reagent(s), generally at 37° C., for a period oftime as indicated for individual studies. Prior to incubation with thereagent, the cells were washed and resuspended in complete media usingthe Sepax® 2 system, using a standard cell washing program and in a kitessentially as shown in FIG. 7. In the same kit, the cells were combinedwith the anti-CD3/28 reagent(s) in the cavity of the chamber by mixingwith intervals of low-speed centrifugation and rest as described forbead incubation for selection for 30 minutes at room temperature.Following the incubation, the incubated material was transferred via theSepax® 2 unit into an output cell culture bag, which then was incubatedat 37° C. for the remainder of the activation period.

6. Transduction

Transduction was carried out in the centrifugal chamber integral to thekit, placed in association with the Sepax® processing unit, as describedin Example 7, with specific details given in particular examples.

7. Expansion

In some cases, following transduction, cells were further incubated,generally at 37 degrees C., to allow for expansion.

8. Wash, Formulation

In some cases, the expanded and/or transduced cells were further washed,diluted, and/or formulated for testing, storage, and/or administration.In some examples, expanded and/or transduced cells were washed in thechamber integral to a single-use kit for use with the Sepax® 2 system,for example as described for cryopreservation. In some cases, a bagcontaining washed cells was sterilely connected to a kit such as shownin FIG. 7, or such a kit with a plurality of ports available forconnection of containers, e.g., bags, at the Output Bag position shownin FIG. 7.

One example of such a multi-port output kit is shown in FIG. 11, whichshows a plurality of ports (17), to one or more of which may beconnected a container, such as a bag, for collection of outputcomposition. The connection may be by sterile welding of the desirednumber of containers, depending for example, on the desired number ofunit dosage form of the cells to produce by a given method. To generatethe kit shown in FIG. 11, a multi-way tubing manifold with a pluralityof ports (in the example shown in FIG. 11, eight) was sterilely weldedto an output line of a single-use kit sold by Biosafe AS forregenerative medicine use. A desired number of plurality of output bagswere sterilely connected to one or more, generally two or more, of theseports. In some examples, such bags were attached to fewer than all theports. Clamps (5) were placed on the tubing lines preventing movement offluid into the individual bags until desired. A bag containing thedesired liquid, such as formulation, assay, and/or cryopreservationmedia, was sterilely connected to the kit and a dilution protocol run onthe Sepax® 2 unit a plurality of times, with the user opening andclosing the respective clamps leading to the appropriate number of bags,thereby generating an output composition in the desired formulation,split into the desired number of bags. In some embodiments, a singleunit dose of cells was collected in each of the respected bags, in aformulation for administration to a subject, such as the subject fromwhich the leukapheresis product was derived.

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

The invention claimed is:
 1. A transduction method, the methodcomprising incubating, in an internal cavity of a centrifugal chamber,an input composition comprising cells and viral particles containing arecombinant viral vector, wherein: the centrifugal chamber comprises: anend wall, a substantially rigid side wall extending from said end wall,and one or more opening, wherein at least a portion of said side wallsurrounds said internal cavity and at least one of the one or moreopening is capable of permitting intake of liquid or gas into saidinternal cavity and expression of liquid or gas from said cavity; and amovable member capable of moving within the chamber to vary the internalvolume of the internal cavity, whereby the internal cavity is a cavityof variable volume defined by said end wall, said substantially rigidside wall, and said movable member; the centrifugal chamber is rotatablearound an axis of rotation and is rotating around said axis of rotationduring at least a portion of the incubation; during at least a portionof the incubation in the chamber or during the rotation of the chamber,the liquid volume of the input composition occupies only a portion ofthe volume of the internal cavity of the chamber, the volume of thecavity during said at least a portion or during said rotation furthercomprising a gas; wherein prior to or during said incubation, effectingmovement of the movable member and effecting intake of the gas into saidinternal cavity, thereby increasing the volume of the internal cavity ofthe chamber; and wherein the method generates an output compositioncomprising a plurality of the cells transduced with the viral vector. 2.The method of claim 1, wherein said rotating comprises rotation at arelative centrifugal force at an internal surface of the side wall ofthe cavity and/or at a surface layer of the cells that is or is at leastat or about 600 g.
 3. The method of claim 1, wherein the at least aportion of the incubation during which the chamber is rotating is for atime that is between about 5 minutes and about 60 minutes, inclusive. 4.The method of claim 1, wherein: the average liquid volume of said inputcomposition present in said cavity during said incubation is no morethan about 5 milliliters (mL) per square inch of the internal surfacearea of the cavity during said incubation.
 5. The method of claim 1,wherein: the number of said cells in said input composition is at orabout the number of said cells sufficient to form a monolayer on thesurface of said cavity during rotation of said centrifugal chamber at aforce of at or about 1000 g or at or about 2000 g at an internal surfaceof the side wall and/or at a surface layer of the cells; and/or thenumber of said cells in said input composition is no more than 2 timesthe number of said cells sufficient to form a monolayer on the surfaceof said cavity during rotation of said centrifugal chamber at a force ofat or about 1000 g or at or about 2000 g at an internal surface of theside wall and/or at a surface layer of the cells; and/or said inputcomposition in the cavity comprises at least at or about 1×10⁶ of saidcells.
 6. The method of claim 1, wherein said input compositioncomprises at least at or about 1 infectious unit (IU) of viral particlesper one of said cells.
 7. The method of claim 1, wherein: the internalcavity of the centrifugal chamber has an internal surface area of atleast at or about 1×10⁹ μm²; the number of cells in the inputcomposition is at least 1×10⁷ cells, and the viral particles are presentin the input composition at, at about, or at least 1 infectious unit(IU) per one of said cells.
 8. The method of claim 7, wherein: theliquid volume of the input composition is less than or equal to 200 mL;and/or the liquid volume of the input composition is no more than 50% ofthe volume of the internal cavity during rotation.
 9. The method ofclaim 7, wherein the volume of said gas is up to 200 mL.
 10. The methodof claim 7, wherein said rotation is at a relative centrifugal force atan internal surface of the side wall of the cavity or at a surface layerof the cells of at least at or about 600 g.
 11. The method of claim 1,wherein: the input composition comprises greater than or about 20 mL,and/or said input composition comprises at least 1×10⁸ cells; and saidrotating conditions comprise a relative centrifugal force on a surfacelayer of the cells of greater than about 800 g.
 12. The method of claim11, wherein said incubation is carried out in a cavity of thecentrifugal chamber and the number of said cells in said inputcomposition is at or about the number of said cells sufficient to form amonolayer or a bilayer on the inner surface of said cavity during saidrotation.
 13. The method of claim 1, wherein a further portion of theincubation of the input composition is carried out outside of thecentrifugal chamber and/or without rotation, said further portioncarried out subsequent to the at least a portion carried out in thechamber and/or with rotation.
 14. The method of claim 13, wherein: thefurther incubation is carried out for a time that is no more than 24hours; the cells in the input composition have not been subjected to atemperature of greater than 30° C. for more than 24 hours; and/or thefurther incubation is not performed in the presence of a stimulatingagent.
 15. The method of claim 1, wherein: the output compositioncontaining transduced cells comprises at least at or about 1×10⁷ cells;for an input composition comprising a virus at a ratio of about 1 orabout 2 IU per cells, said method is capable of producing an outputcomposition in which at least 10% of the cells in said outputcomposition generated by the method comprise said recombinant viralvector and/or express a product of a recombinant nucleic acid comprisedwithin said vector; or among all the cells in said output compositionthat contain the recombinant viral vector or into which the viral vectoris integrated, the average copy number of said recombinant viral vectoris no more than about 10; or among the cells in the output composition,the average copy number of said vector is no more than about
 2. 16. Themethod of claim 1, wherein the centrifugal chamber is integral to aclosed system, said closed system comprising: said chamber and at leastone tubing line operably linked to at least one of the one or moreopening via at least one connector, whereby liquid and gas are permittedto move between said cavity and said at least one tubing line in atleast one configuration of said closed system; and at least onecontainer operably linked to the at least one tubing line, theconnection permitting liquid and/or gas to pass between said at leastone container and at least one of the one or more opening via the atleast one tubing line.
 17. The method of claim 16, wherein said at leastone container comprises at least one input container comprising saidviral vector particles and said cells, a waste container, a productcontainer, and at least one diluent container, and said at least onetubing line comprises a series of tubing lines, wherein each of saidcontainers is connected to said cavity via said series of tubing linesand at least one of the one or more opening.
 18. The method of claim 17,wherein said method further comprises, prior to and/or during saidincubation, effecting intake of said input composition into said cavity,said intake comprising flowing of liquid from said at least one inputcontainer into said cavity through at least one of the one or moreopening; and/or prior to and/or during said incubation, providing oreffecting intake of said gas into said cavity under sterile conditions,said intake being effected by (a) flow of said gas from a container thatcomprises said gas, (b) flow of said gas from an environment external tothe closed system, via a microbial filter, or (c) flow of said gas froma syringe connected to the closed system at a syringe port.
 19. Themethod of claim 17, wherein the closed system comprises: at least onefurther container that comprises said gas for intake into the internalcavity prior to and/or during at least a point during said incubation;and/or a microbial filter capable of taking in gas to the internalcavity of the centrifugal chamber; and/or a syringe port for effectingintake of said gas.
 20. The method of claim 18, wherein: the effectingintake of the gas into the internal cavity of the centrifugal chamber iscarried out simultaneously or together with the effecting intake of theinput composition to the internal cavity of the centrifugal chamber; orthe effecting of the intake of the gas is carried out separately, eithersimultaneously or sequentially, from the effecting of the intake of theinput composition into said cavity.
 21. The method of claim 1, whereinthe gas is air.
 22. The method of claim 1, wherein the incubation ispart of a continuous process, the method further comprising: during atleast a portion of said incubation, effecting continuous intake of saidinput composition into said cavity during rotation of the chamber; andduring a portion of said incubation, effecting continuous expression ofliquid from said cavity through at least one of the one or more openingduring rotation of the chamber.
 23. The method of claim 1, wherein theincubation is part of a semi-continuous process and the method furthercomprises: prior to said incubation, effecting intake of said inputcomposition, and said gas, into said cavity through at least one of theone or more opening; subsequent to said incubation, effecting expressionof liquid and/or said gas from said cavity; effecting intake of anotherinput composition comprising cells and said viral particles containing arecombinant viral vector, and gas, into said internal cavity; andincubating said another input composition in said internal cavity,wherein the method generates another output composition comprising aplurality of cells of the another input composition that are transducedwith said viral vector.
 24. The method of claim 18, wherein the methodfurther comprises: effecting rotation of said centrifugal chamber priorto and/or during said incubation; effecting expression of liquid fromsaid cavity into said waste container following said incubation;effecting expression of liquid from said at least one diluent containerinto said cavity via at least one of the one or more opening andeffecting mixing of the contents of said cavity; and effectingexpression of liquid from said cavity into said product container,thereby transferring cells transduced with the viral vector into saidproduct container.
 25. The method of claim 1, further comprising: (a)washing a biological sample comprising said cells in an internal cavityof the centrifugal chamber prior to said incubation; and/or (b)isolating said cells from a biological sample, wherein at least aportion of the isolation step is performed in an internal cavity of thecentrifugal chamber prior to said incubation; and/or (c) stimulatingcells prior to and/or during said incubation, said stimulatingcomprising exposing said cells to stimulating conditions, therebyinducing cells of the input composition to proliferate, wherein at leasta portion of the step of stimulating cells is performed in an internalcavity of the centrifugal chamber.
 26. The method of claim 1, wherein:said cells in said input composition comprise suspension cells; saidcells in said input composition comprise white blood cells; and/or saidcells in said input composition comprise T cells or NK cells.
 27. Themethod of claim 1, wherein during said incubation, said centrifugalchamber is located within a centrifuge and associated with a sensor,said sensor capable of monitoring the position of said movable member,and control circuitry capable of receiving and transmitting informationfrom said sensor and causing movement of said movable member, intake andexpression of liquid and/or gas to and from said cavity via one or moretubing lines, and rotation of said chamber via said centrifuge.
 28. Themethod of claim 1, wherein said recombinant viral vector encodes arecombinant receptor, which is thereby expressed by cells of the outputcomposition.
 29. The method of claim 1, wherein: the movable member is apiston; and/or the movable member is capable of axially moving withinthe chamber to vary the internal volume of the cavity.
 30. The method ofclaim 1, wherein: the maximum total liquid volume of said inputcomposition present in said cavity at any one time during saidincubation is no more than 100 times the total volume of said cells insaid cavity or the average liquid volume of the input composition overthe course of the incubation is no more than 100 times the total volumeof cells in the cavity, or the liquid volume of the input composition isno more than 200 mL.
 31. The method of claim 1, further comprisingformulating cells transduced by the method in a pharmaceuticallyacceptable buffer in an internal cavity of the centrifugal chamber,thereby producing a formulated composition.
 32. The method of claim 31,further comprising effecting expression of the formulated composition toone or a plurality of containers.
 33. The method of claim 1, whereinsaid movement of said movable member is axial within said centrifugalchamber.
 34. The method of claim 1, wherein said method comprises priorto said incubation, effecting intake of said input composition into saidinternal cavity through at least one of the one or more opening.
 35. Themethod of claim 1, wherein the intake of said gas into said internalcavity is carried out under sterile conditions.
 36. The method of claim35, wherein the centrifugal chamber is integral to a closed system, andwherein the intake of gas is effected by (a) flow of said gas from acontainer that comprises said gas, (b) flow of said gas from anenvironment external to the closed system, via a microbial filter, or(c) flow of said gas from a syringe connected to the closed system at asyringe port.
 37. The transduction method of claim 1, wherein movementof the movable member effects the intake of said gas into the internalcavity.
 38. The transduction method of claim 35, wherein movement of themovable member effects the intake of said gas into the internal cavity.39. The transduction method of claim 1, wherein the maximum liquidvolume of said input composition present in said cavity at any one timeduring said incubation is no more than about 5 mL per square inch of themaximum internal surface area of the cavity.
 40. The transduction methodof claim 1, wherein the maximum liquid volume of said input compositionpresent in said cavity at any one time during said incubation, or theaverage liquid volume over the course of the incubation, is no more thanat or about 2 times the volume of a monolayer of said cells formed onthe inner surface of said cavity during rotation of said chamber at aforce of at or about 1000 g or at or about 2000 g at an internal surfaceof the side wall and/or at a surface layer of the cells.
 41. The methodof claim 1, wherein: the internal cavity of the centrifugal chamber hasan internal surface area of at least at or about 1×10¹⁰ μm²; the numberof cells in the input composition is at least 1×10⁷ cells, and the viralparticles are present in the input composition at, at about, or at least1 infectious unit (IU) per one of said cells.
 42. The method of claim 1,wherein the liquid volume of said input composition present in saidcavity during rotation of said centrifugal chamber per square inch ofthe internal surface area of the cavity is decreased compared to theabsence of gas in the chamber.